Wiki FKKT - User contributions [en]

Potenciali interakcij med algami in bakterijami.

2015-06-10T07:05:37Z

MatjaZalar: New page: =UVOD= Alge so fototrofni evkarionti, ki se večinoma nahajajo v vodnih ekosistemih. Rezultati ekoloških študij so pokazali, da posamezne skupine bakterij najdemo samo v bližini specifi...

=UVOD=
Alge so fototrofni evkarionti, ki se večinoma nahajajo v vodnih ekosistemih. Rezultati ekoloških študij so pokazali, da posamezne skupine bakterij najdemo samo v bližini specifičnih vrst alg. Kolonije bakterij tvorijo nekakšen biofilm na površini alg, ki pa lahko vključuje tudi druge organizme. Take združbe alg in heterotrofov imenujemo fikosfera (angl. phycosphere), kjer alge predstavljajo vir energije za heterotrofe, ki jih naseljujejo. Dokazano pa je tudi, da fikosferske bakterije vplivajo na rast, morfogenezo in kolonizacijo alg. Interakcije med algami in bakterijami so tako ključnega pomena pri oblikovanju in ohranjanju vodnih ekosistemov. Poleg tega pa so njihove interakcije zanimive tudi iz biotehnološkega vidika. Uporabljali bi jih lahko za čiščenje odpadnih vod, proizvodnjo biogoriva in, kot se kaže v zadnjem času, za biogorivne celice.
=VRSTE INTERAKCIJ MED ALGAMI IN BAKTERIJAMI=
Interakcije med algami in bakterijami delimo v tri skupine: izmenjavo hranil, prenos signalov in prenos genov.
'''Izmenjava hranil''': Izmenjava hranil je najpogostejša oblika interakcij med algami in bakterijami. Alge del fotosintetsko proizvedenih snovi izločijo v obliki raztopljenega organskega ogljika (angl. dissolved organic carbon oz. DOC), le te pa v veliki meri asimilirajo in razgradijo heterotrofne bakterije. Sestava DOC določa tudi vrste bakterij, ki uspevajo na površini alg. Poleg tega odmrle in obolele alge predstavljajo dodaten vir energije za heterotrofe v njihovi bližini. Primerjava kolonizacije bakterij na zdravih in bolnih makroalgah je pokazala, da so zdrave alge sposobne nadzorovati kolonizacijo bakterij na njihovi površini medtem ko obolele alge te funkcije nimajo. Poseben primer interakcij je endosimbioza cianobakterij in evkarionstakih alg, ki je ključnega pomena pri fiksaciji dušika iz okolja v vodnih ekosistemih. Obstajajo celo dokazi, da določene bakterije posredujejo pri asimilaciji dušika v alge, ter tako pospešijo njihovo rast. Nekatere skupine alg izločajo prosojne ekskopolimerne delce (TEP, anlg. transparant exopolymer particles), ki določajo vrsto bakterij v fikosferi ter vplivajo na njihovo aktivnost.
'''Prenos signalov:''' Obojestranski prenos signalov med algami in bakterijami poteka preko signalnih molekul, ki aktivirajo ali inhibirajo izražanje genov ter vplivajo na fiziološke procese kot sta rast in obnašanje. Alge nadzorujejo rast bakterij preko inhibitornih molekul, ki vplivajo na quorum sensing bakterij, zaradi česar niso sposobne tvorbe biofilma. Morske alge izločajo hlapne halogenirane spojine in maščobne kisline, ki delujejo baktericidno. Po drugi strani pa družine bakterij kot so ''Shewanella, Streptomyces'' in ''Bacillus'', izločajo algicidne metabolite. Poleg tega so znani metaboliti bakterij, ki inducirajo morfogenezo alg. Prenos signalov med algami in bakterijami bi se lahko uporabljal za nadzorovanje in izboljševanje biotehnoloških procesov.
'''Prenos genov:''' Študije posameznih fikosfer nakazujejo na horizontalni prenos genov med algami in bakterijami. Genom diatomej med drugim vsebuje zapis za encime ciklusa uree, ki so se nanje predvidoma prenesli iz bakterij, ter bistveno izboljšali algino sposobnost izrabe občasno razpoložljivega dušika iz okolja.
=TEHNOLOŠKE APLIKACIJE=
Združbe med algami in bakterijami lahko vključimo v postopke čiščenja odpadnih vod ter proizvodnje biogoriva. Pri proizvodnji biogoriva je uporaba bakterij omejena, saj razgrajujejo željene sintetizirane snovi kot so olja, vodik, idr., kar vodi v nižje izkoristke proizvodnje. Hkrati pa je znano, da nekatere bakterije povzročijo agregacijo celic alg, kar omogoča hitrejšo in učinkovitejšo proizvodnjo biogoriva.
Alge se že uporablja pri čiščenju umazanih odpadnih vod, vendar se z odkrivanjem potencialov interakcij med algami in bakterijami ponujajo možnosti napredka že uveljavljenih procesov. Pri študiji čiščenja odpadnih vod z algo ''Chlorella vulgaris'', se je izkazalo, da je dodatek bakterije ''Azospirillum brasilense'' bistveno izboljšal delovanje alg in pohitril celoten proces. Poleg tega so znane tudi bakterije, ki zavirajo rast alg in bi jih lahko uporabljali pri zaustavljanju neželjenega pojava cvetenja alg.
Zadnji primer uporabe interakcij med algami in bakterijami so samo obnavljajoče biološki sistemi za pretvorbo svetlobe v elektriko (angl. self-sustained biological light/electricity-conversion systems) ali krajše MSC. V takem sistemu anodo predstavlja biofilm iz bakterij, ki proizvajajo električni tok ali svetlobo, in alg, ki zagotavljajo zadostno količino hranil. Daleč v prihodnosti bi lahko take biogorivne celice uporabljali kot vir električne energije.
=ZAKLJUČEK=
Zaenkrat je o interakcijah med algami in bakterijami znanega le malo, vendar dovolj da se kaže njihova potencialna uporaba v biotehnoloških procesih, kot so bioremediacija, proizvodnja biogoriva in biogorivne celice.

MBT seminarji 2015

2015-06-10T06:43:14Z

MatjaZalar:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline''' 
# Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015
#A simpliﬁed and accurate detection of the genetically modiﬁed wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]]. Matej Lesar, 20. marca 2015

'''Gensko spremenjene živali''' 
# [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)
# Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015

'''Okolje''' 
# Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). [[Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil]]. Mitja Crček, 3. aprila 2015
# Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). [[Biorazgradnja atrazina s tremi transgenskimi travami in lucerno, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo]]. Mirjam Kmetič, 3. aprila 2015

'''Terapevtiki''' 
# Glycosylated enfuvirtide: A long-lasting glycopeptide with potent anti-HIV activity; http://pubs.acs.org/doi/full/10.1021/jm5016582 [[Glikoliziran Enfuvirtid: glikopeptid z močno proti HIV aktivnostjo s podaljšanim delovanjem]]. Sebastian Pleško, 10. aprila
# Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa; http://ini.sagepub.com/content/21/1/17.long. [[Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne Staphylococcus aureus in Pseudomonas aeruginosa]]. Ana Kapraljević, 10. aprila

'''Encimi''' 
# Immobilization and controlled release of β-galactosidase from chitosan-grafted hydrogels; http://www.sciencedirect.com/science/article/pii/S0308814615001028. [[Imobilizacija in nadzorovano sproščanje β-galaktozidaze iz hitozanskega hidrogela]]. Mojca Banič, 16. aprila 2015
# Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). [[Priprava Pichie pastoris, ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov]]. Špela Tomaž, 17. aprila 2015
# Postharvest application of a novel chitinase cloned from ''Metschnikowia fructicola'' and overexpressed in ''Pichia pastoris'' to control brown rot of peaches; http://www.sciencedirect.com/science/article/pii/S0168160515000033. [[Uporaba hitinaze, klonirane iz Metschnikowie fructicola in prekomerno izražene v Pichii pastoris za nadzor rjave gnilobe breskev po obiranju]] Špela Pohleven, 17. aprila 2015

'''Protitelesa''' 
# Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells; http://dx.plos.org/10.1371/journal.pone.0116878. Optimizacija signalnih peptidov težkih in lahkih verig za večjo ekspresijo terapevtskih protiteles v CHO celičnih linijah. [[Optimizacija signalnih peptidov težkih in lahkih verig za večjo ekspresijo terapevtskih protiteles v CHO celičnih linijah]] Tjaša Blatnik, 23. aprila 2015
# Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). [[Čiščenje rekombinantnih protiteles z obarjanjem z etanolom]]. Urška Rauter, 24. aprila 2015
# Functional mutations in and characterization of VHH against ''Helicobacter pylori'' urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). [[Funkcionalne mutacije in karakterizacija VHH proti ureazi Helicobacter pylori]]. Marko Radojković, 7. maja 2015

'''Cepiva''' 
# Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions; http://www.sciencedirect.com/science/article/pii/S0378517315001507. [[Razvoj pegiliranih lipopleksov proti E6 za aplikacijo na sluznico pri predrakavih spremembah materničnega vratu]]. Tanja Korpar, 7. maja 2015
# A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. [[Nova "priming-boosting" strategija za imunsko posredovanje pri raku materničnega vratu]]. Anita Kustec, 8. maja 2015
# Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) [[Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena.]] Daša Pavc, 8. maja 2015

'''Male molekule in polimeri''' 
# Methanol-induced chain termination in poly(3-hydroxybutyrate) biopolymers: Molecular weight control; http://www.sciencedirect.com/science/article/pii/S0141813014008307. [[Z metanolom inducirana terminacija polimerizacije poli(3-hidroksibutiratnih) polimerov: Vpliv na molekulsko maso]]. Gašper Lavrenčič, 14. maja 2015
# Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20; http://www.sciencedirect.com/science/article/pii/S0141813014008216. Uroš Stupar, 14. maja 2015
# Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases (Citorik RJ. ''et al''; Nature Biotechnology 32, 1141-1145, 2014; http://www.nature.com/nbt/journal/v32/n11/full/nbt.3011.html). [[Sekven%C4%8Dno specifi%C4%8Dna protimikrobna sredstva]] Iza Ogris, 15. maja 2015
# Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) [[Integracija genov za sintezo hialuronske kisline v kromosom bakterije Lactococcus lactis izboljša sintezo visokomolekularne hialuronske kisline]] Maja Grdadolnik, 15. maja 2015

'''Pretvorba biomase''' 
# Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse (L. Jia ''et al''; Bioresource Technology 185, 2015; http://www.sciencedirect.com/science/article/pii/S0960852415002114) [[Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase]]. Eva Lucija Kozak, 21. maja 2015
# Third generation biohydrogen production by Clostridium butyricum and adapted mixed cultures from Scenedesmus obliquus microalga biomass; http://www.sciencedirect.com/science/article/pii/S0016236115002550?np=y [[Tretja generacija proizvodnje biovodika s pomočjo, z mikroalgami Scenedesmus obliquus hranjenimi bakterijami Clostridium butyricum in mešanico prilagojenih mikroorganizmov]] Nives Naraglav, 22. maja 2015
# Bio-catalytic action of twin-screw extruder enzymatic hydrolysis on the deconstruction of annual plant material: Case of sweet corn co-products; http://www.sciencedirect.com/science/article/pii/S0926669015000436 [[Biokatalitični učinek encimske hidrolize dvovijačnega ekstruderja na destrukturiranje rastlinskega materiala]]. Griša Prinčič, 22. maja 2015

'''Metabolično inženirstvo''' 
# Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica (K. Qiao ''et al.''; Metabolic Engineering 29, 2014; http://www.sciencedirect.com/science/article/pii/S1096717615000166) [[Povečanje proizvodnje lipidov v kvasovki Yarrowia lipolytica]]. Andreja Bratovš, 28. maja 2015
# Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). [[Metabolno inženirstvo kvasovke Saccharomyces cerevisiae za proizvodnjo biogoriva in kemikalij iz maščobnih kislin]]. Dominik Kert, 29. maja 2015
# Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). [[Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline]]. Jure Zabret, 29. maja 2015

'''Biološki viri energije''' 
# Anodic and cathodic microbial communities in single chamber microbial fuel cells; http://www.sciencedirect.com/science/article/pii/S1871678414021694. [[Anodna in katodna mikrobna združba v eno-celični mikrobni gorivni celici]] Tamara Marić, 4. junija 2015
# Combination of dry dark fermentation and mechanical pretreatment for lignocellulosic deconstruction: An innovative strategy for biofuels and volatile fatty acids recovery; http://www.sciencedirect.com/science/article/pii/S0306261915002196. [[Kombinacija temne fermentacije v trdnem stanju in mehanske obdelave za razgradnjo lignoceluloze: Inovativen pristop za proizvodnjo biogoriv in hlapnih organskih kislin.]] Jernej Pušnik, 4. junija 2015
# Potential use of feedlot cattle manure for bioethanol production; http://www.sciencedirect.com/science/article/pii/S0960852415001960. [[Uporaba govejega gnoja v proizvodnji bioetanola.]] Nastja Pirman, 5. junija 2015
# Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production; http://www.sciencedirect.com/science/article/pii/S0960148114007022. [[Celulolitični encimi talne glive Penicillium sp. TG2 in njihov potencial pri proizvodnji etanola.]] Jana Verbančič, 5. junija 2015

'''Novi pristopi v molekularni biotehnologiji''' 
# Exploring the potential of algae/bacteria interactions; http://www.sciencedirect.com/science/article/pii/S0958166915000269. [[Potenciali interakcij med algami in bakterijami.]] Matja Zalar, 11. junija
# How close we are to achieving commercially viable large-scale photobiological hydrogen production by cyanobacteria: A review of the biological aspects; http://www.mdpi.com/2075-1729/5/1/997/htm. [[Kako blizu smo dosegu komercialno dostopne masovne fotobiološke proizvodnje vodika z cianobakterijam: pregled z biološkega vidika.]] Monika Škrjanc, 11. junija
# Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) [[Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka.]] Luka Smole, 11. junija 2015

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-19T07:52:32Z

MatjaZalar: /* Design of CCR5 ZFNs */

Perez, E. E. ''et al.'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/pdf/nihms-177273.pdf Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases]. Nat Biotechnol, 26, 808-16 (2008).

==Introduction==
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ''ex vivo'' and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps:
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules as it is shown in [http://www.nature.com/nrd/journal/v2/n5/fig_tab/nrd1087_F1.html FIG 1] This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain.

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal.

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp120. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>

Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function. Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype ''de novo''. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out ''in vitro'' as well as ''in vivo'' in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleavage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleavage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specificity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data.

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection ''in vivo''.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out ''in vitro'' as well as ''in vivo''. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.

==References==
<references/>

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-19T07:52:11Z

MatjaZalar: /* Design of CCR5 ZFNs */

Perez, E. E. ''et al.'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/pdf/nihms-177273.pdf Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases]. Nat Biotechnol, 26, 808-16 (2008).

==Introduction==
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ''ex vivo'' and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps:
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules as it is shown in [http://www.nature.com/nrd/journal/v2/n5/fig_tab/nrd1087_F1.html FIG 1] This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain.

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal.

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp120. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>

Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function. Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype ''de novo''. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out ''in vitro'' as well as ''in vivo'' in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleavage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleveage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specificity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data.

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection ''in vivo''.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out ''in vitro'' as well as ''in vivo''. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.

==References==
<references/>

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-19T07:39:37Z

MatjaZalar: /* HIV-1 co-receptors */

Perez, E. E. ''et al.'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/pdf/nihms-177273.pdf Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases]. Nat Biotechnol, 26, 808-16 (2008).

==Introduction==
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ''ex vivo'' and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps:
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules as it is shown in [http://www.nature.com/nrd/journal/v2/n5/fig_tab/nrd1087_F1.html FIG 1] This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain.

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal.

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp120. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>

Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function. Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype ''de novo''. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out ''in vitro'' as well as ''in vivo'' in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleveage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleveage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specificity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data.

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection ''in vivo''.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out ''in vitro'' as well as ''in vivo''. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.

==References==
<references/>

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-18T22:49:16Z

MatjaZalar:

Perez, E. E. ''et al.'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/pdf/nihms-177273.pdf Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases]. Nat Biotechnol, 26, 808-16 (2008).

==Introduction==
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ''ex vivo'' and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps:
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules as it is shown in [http://www.nature.com/nrd/journal/v2/n5/fig_tab/nrd1087_F1.html FIG 1] This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain.

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal.

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp41. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>

Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function. Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype ''de novo''. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out ''in vitro'' as well as ''in vivo'' in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleveage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleveage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specificity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data.

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection ''in vivo''.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out ''in vitro'' as well as ''in vivo''. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.

==References==
<references/>

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-18T22:40:46Z

MatjaZalar:

Perez, E. E. "et al." [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/pdf/nihms-177273.pdf Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases]. Nat Biotechnol, 26, 808-16 (2008).

==Introduction==
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ex vivo and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps:
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules as it is shown in [http://www.nature.com/nrd/journal/v2/n5/fig_tab/nrd1087_F1.html FIG 1] This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain.

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal.

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp41. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>

Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function. Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype de novo. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out in vitro as well as in vivo in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleveage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleveage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specifity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data.

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection in vivo.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out in vitro as well as in vivo. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.

==References==
<references/>

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-18T22:36:32Z

MatjaZalar: /* Zinc finger domain */

==Introduction==
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ex vivo and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps:
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules as it is shown in [http://www.nature.com/nrd/journal/v2/n5/fig_tab/nrd1087_F1.html FIG 1] This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain.

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal.

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp41. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>

Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function. Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype de novo. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out in vitro as well as in vivo in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleveage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleveage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specifity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data.

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection in vivo.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out in vitro as well as in vivo. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.

==References==
<references/>

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-18T22:34:57Z

MatjaZalar: /* Zinc finger domain */

==Introduction==
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ex vivo and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps:
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules as it is shown in [http://www.nature.com/nrd/journal/v2/n5/fig_tab/nrd1087_F1.html. FIG 1] This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain.

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal.

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp41. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>

Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function. Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype de novo. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out in vitro as well as in vivo in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleveage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleveage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specifity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data.

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection in vivo.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out in vitro as well as in vivo. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.

==References==
<references/>

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-18T22:34:06Z

MatjaZalar: /* Zinc finger domain */

==Introduction==
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ex vivo and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps:
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules as it is shown [http://www.nature.com/nrd/journal/v2/n5/fig_tab/nrd1087_F1.html. FIG 1] This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain.

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal.

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp41. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>

Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function. Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype de novo. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out in vitro as well as in vivo in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleveage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleveage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specifity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data.

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection in vivo.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out in vitro as well as in vivo. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.

==References==
<references/>

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-18T22:28:36Z

MatjaZalar:

==Introduction==
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ex vivo and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps:
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules. This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain.

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal.

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp41. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>

Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function. Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype de novo. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out in vitro as well as in vivo in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleveage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleveage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specifity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data.

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection in vivo.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out in vitro as well as in vivo. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.

==References==
<references/>

Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases

2015-01-18T21:26:12Z

MatjaZalar: New page: ==Introduction== In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 differen...

SB students resources

2015-01-18T21:24:23Z

MatjaZalar: /* List of articles for presentation */

===Introduction to our students resources in Synthetic Biology===
(Marko Dolinar)

Synthetic biology made a vast progress in good 10 years since it established itself as an interdisciplinary field of research on the interface of molecular biology and engineering. University of Ljubljana Faculty of Chemistry and Chemical Technology has introduced a Synthetic Biology course as a part od Biochemistry MSc programme only in 2013/14. This is relatively late, considering a great success of Slovenian students at iGEM competitions since their first attendance in 2006. On the other hand, the field is still in its first stages if development and a complete textbook for a MSc level course is still missing. This is the reason why our students collaborated on the preparation of a Synthetic Biology textbook with the working title Synthetic Biology - A Students Textbook. It exists as a draft that is not publicly available and is actually part 1 of a (to be) 2-volumes title. Part I is subtitled Engineering Biology, while Part II (that currently doesn't exisist yet) will be subtitled Synthetic Biology Applications.

As in all highly competitive fields of science and technology, students should be following recent progress by reading articles in high quality journals. However, this is often a very difficult task, especially at the BSc level. Specificities of the scientific and technical language, push of publishers towards very short methodological chapters and limited knowledge studens might have about advanced techniques make understanding papers a very challenging task. Therefore, I decided to face MSc students with the challenge to explain selected SB articles in a manner that would make the content of these articles understandable to BSc level students and non-experts.

In 2014/15, seminars in Synthetic Biology include explanations and presentations of some of the top-cited articles from the field of Synthetic Biology. I compiled a list of 95 articles published between 2000 and 2014 having the highest number of citations according to the Web of Science database. The list ended with the paper just exceeding the 100 citations limit. Not included in the list were reviews. With 20 students enrolled in the course, the list has been further reduced to top 40 papers in the field. Students have been asked to check for content (they further eliminated 3 papers which proved to be reviews) and availabitly (they all seemed to be available as full texts with our university subscriptions). My suggestion was to avoid selecting for presentation papers with very similar content. Especially in the field of genome editing there has been a very rapid progress in the past few years resulting in a number of highly-cited articles which could appear very similar in content for a non-specialist. From the shortlist of 37 articles, students selected a topic they believed would be most interesting or easiest to explain. Presentations will be both written (in English, which is not the mother tongue of my students) and oral (in Slovenian, to establish and maintain Slovenian terminology in the field).

===List of articles for presentation===

This is the list of top-cited papers from the broader field of Synthetic Biology that students chose for explanation in 2014/15 (sorted by year of publication):

#[[A synthetic oscillatory network of transcriptional regulators]], Michael B. Elowitz & Stanislas Leibler, Letters to Nature, 2000 - Valter Bergant
#[[Construction of a genetic toggle switch in Escherichia coli]]. Gardner ''et al''., Nature, 2000 - Urban Bezeljak
#Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion (2001) - Andreja Bratovš
#Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template (2002) - Veronika Jarc
#[[Combinatorial synthesis of genetic networks]]. Guet C.C. ''et al'', Science, 2002 - Maja Remškar
#Engineering a mevalonate pathway in Escherichia coli for production of terpenoids (2003) - Ana Kapraljević
#Programmed population control by cell-cell communication and regulated killing. You et al, Nature (2004)[http://wiki.fkkt.uni-lj.si/index.php/7.Programmed_population_control_by_cell-cell_communication_and_regulated_killing] - Alja Zottel
#Gene regulation at the single-cell level (2005) - Katarina Uršič
#[[A synthetic multicellular system for programmed pattern formation]]. (2005) - Mitja Crček
#[[Long-term monitoring of bacteria undergoing programmed population control in a microchemostat]]. Balagadde ''et al.'', ''Science'', 2005 - Jana Verbančič
#[[Tuning genetic control through promoter engineering]], Hal Alper ''et al''., PNAS, 2005 - Špela Pohleven
#Production of the antimalarial drug precursor artemisinic acid in engineered yeast (2006) - Živa Marsetič
#[[An improved zinc-finger nuclease architecture for highly specific genome editing]], Miller ''et al''., ''Nature Biotechnol''., 2007 - Eva Knapič
#[[Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases]] (2008) - Tamara Marić
#[[Synthetic protein scaffolds provide modular control over metabolic flux]]. Dueber ''et al''., Nature Biotechnology, 2009. - Ana Dolinar
#[[Creation of a bacterial cell controlled by a chemically synthesized genome]]. Gibson, D. G. ''et al.'', Science, 2010 - Eva Lucija Kozak
#[[A TALE nuclease architecture for efficient genome editing]], Miller ''et al'', ''Nature Biotechnol''., 2011 - Jernej Mustar
#Multiplex genome engineering using CRISPR/Cas systems (2013) - Uroš Stupar
#[[RNA-guided human genome engineering via Cas9]]. Mali ''et al''., Science, 2013 - Luka Smole
#[[One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering (2013)]] - Andrej Vrankar

''Please link the title of each paper with your written seminar wiki page. Expand the citation according to the following example:
''
#Emergent bistability by a growth-modulating positive feedback circuit. Tan et al., Nature Chem. Biol., 2009

Regulation of flowering time in Arabidopsis thaliana

2014-01-18T12:06:14Z

MatjaZalar: /* ČLANEK */

=='''UVOD'''==
Cvetenje (angl. ‘flowering’) je kompleksno uravnan rastlinski proces, ki se prične kot odgovor na različne okoljske in endogene dejavnike. Znanstveniki so z genetskimi študijami določili štiri glavne skupine, ki kontrolirajo proces cvetenja: vernalizacijska pot, fotoperiodična pot, avtonomna in giberelinska pot. Znanstveniki so identificirali veliko genov, ki regulirajo čas cvetenja (angl. ‘flowering time’) v ''Arabidopsis thailana'' in se medsebojno povezujejo. Mutacije v tovrstnih genih se fenotipsko odražajo v zakasnitvi cvetenja. V mnogih vrstah rastlin je za to potreben prehod iz juvenilne v odraslo fazo, ki pa je reguliran preko avtonomne poti.
Namen raziskave je bila določitev pomembnosti dveh transkripcijskih faktorjev (TF), atVOZ1 in atVOZ2 pri razvoju in cvetenju rastline ''Arabidopsis thailana''.

=='''METODE IN REZULTATI'''==
V raziskavi so uporabili rastlino A. thaliana in mutantne linije znotraj enakega ekotipa. Poskuse so delali na dveh enojnih mutantah (atVOZ1-1 in atVOZ2-1), na dvojni mutanti (atVOZ1-1 atVOZ2-1) in na dvojni mutanti, ki je bila komplementirana z atVOZ2 (atVOZ2; atVOZ1-1 atVOZ2-1) ter jih primerjali z divjim tipom. . V začetnih eksperimentih so opazovali fenotip vseh mutantov ter jih primerjali z divjim sevom. Merili so čas cvetenja, ki je predstavljal čas od dneva saditve do prvega cveta in število rozetnih lističev. Čas cvetenja dvojnih mutant je daljši kot pri divjem sevu, medtem ko v primeru enojnih mutant velikega odstopanja od divjega seva ni bilo, a je bilo še vedno statistično signifikantno. Število rozetnih lističev je bilo pri dvojnih mutantah v DD več kot pri divjem sevu.
Naslednja stopnja poskusa je bila potrditev rezultatov, pridobljenih z opazovanjem fenotipa, na molekularni ravni, z različnimi molekularnimi tehnikami. Z metodo RT-PCR so analizirali ekspresijo genov v vseh mutantah in jih primerjali z divjim tipom, ki so bili že iz prejšnjih raziskav znani, da regulirajo proces cvetenja. Flowering locus C (FLC) je gen, ki kodira protein s funkcijo represije cvetenja in le-ta je bil nadizražen v dvojnih mutantah in prav tako velja za preostale preiskovane regulatorje FLC-ja. Gena FD in FT kodirata proteina, ki promovirata cvetenje. V dvojnih mutantah je bil FD podizražen, medtem ko za FT ni bilo opaznih sprememb. S tem eksperimentom so potrdili značilni fenotip zaostalega cvetenja dvojnih mutant, saj so geni, ki so vključeni v represijo tega procesa nadizraženi, aktivatorji pa so podizraženi.
Znanstvenike je zanimala odzivnost dvojnih mutant po tem, ko so bila semena dvanajst tednov izpostavljena vernalizacijskim pogojem. Opazovali so fenotip, torej cvetni čas in število rozetnih listov ter izražanje gena FLC divjega tipa in dvojnih mutant pri rastlinah, ki so bile izpostavljene pogojem vernalizacije in tistim, ki niso bile. Opazili so, da se je v dvojnih mutantah nivo transkripta gena FLC znižal in rastline so začele cveteti mnogo prej kot dvojne mutante v nevernatalizacijskih pogojih.
Znanstveniki so želeli preučiti interakcije atVOZ proteinov s specifičnimi promotorji genov, ki so bili že predmet raziskav tekom te študije. Ustreznega zaporedja, znanega tudi kot vezavo mesto za VOZ, niso našli v nobeni promotorski regiji, niti gena FLC, niti njegovih regulatorjev kar nakazuje, da ni direktne povezave med TF-jema in prej opisanimi geni. Povezava pa je lahko posredna, zato so iskanje vezavega mesta za VOZ razširili na 1000 bp navzgor od promotorskih zaporedij. Našli so ustrezno vezavno mesto na promotorju gena Modifier of SNC1,3/Supressor of auxin resistance 3 (MOS3/SAR3), ki kodira jedrni membranski protein. V tem eksperimentu so znanstveniki želeli preučiti nivo izražanja MOS3/SAR3 v dvojnih mutantah ter preučiti interakcije atVOZ proteinov z DNA zaporedjem gena MOS3/SAR3 s pomočjo testa zamika elektroforezne mobilnosti (EMSA). Ugotovili so, da je gen MOS3/SAR3 nadizražen in da je vezava atVOZ2 na promotorsko regijo MOS3/SAR3 odvisna od koncentracije proteina. Poleg tega pa so tudi dokazali, da se atVOZ2 na vezavno mesto za VOZ veže s Zn2+ vezavno domeno in tako regulira izražanje gena MOS3/SAR3. To so prav tako določili z metodo EMSA, le da so opazovali vezavo mutiranega atVOZ2 proteina, z mutacijami narejenimi v vezavni domeni, na promotorsko regijo ter vezavo atVOZ2 na vezavnem mesto za VOZ, znotraj katerega je bila narejena mutacija. Zaradi težav pri izražanju atVOZ1 proteina tega zanj niso mogli dokazati, ampak so do podobnih rezultatov prišli z metodo kromatinske imunoprecipitacije (ChIP).

=='''ZAKLJUČEK'''==
Raziskava potrjuje pomembno vlogo transkripcijskih faktorjev atVOZ1 in atVOZ2 v regulaciji prehoda iz vegetativne rasti v cvetenje. Z različnimi eksperimenti, ki si sledijo v logičnem zaporedju so dokazali, da imajo rastline (''atVOZ1-1 atVOZ2-1'') daljši čas cvetenja, kadar le-tega primerjamo z divjim sevom. Dvojni mutanti so občutljivi na vernalizacijo kar se vidi v fenotipu, ki je podoben divjemu sevu in v znižanju izražanja gena FLC. Prav tako pa so dokazali, da transkripcijska faktorja posredno vplivata na gene, ki sodelujejo pri prehodu iz vegetativne rasti v cvetenje, in zagotovo ima del vloge pri tem gen MOS3/SAR3, na katerega transkripcijska faktorja delujeta neposredno.
=='''ČLANEK'''==
CELESNIK, H., ALI, G. S., ROBISON, F. M. & REDDY, A. S. 2013. Arabidopsis thaliana VOZ (Vascular plant One-Zinc finger) transcription factors are required for proper regulation of flowering time. Biol Open, 2, 424-31. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3625871/

Regulation of flowering time in Arabidopsis thaliana

2014-01-18T12:03:59Z

MatjaZalar: /* UVOD */

Regulation of flowering time in Arabidopsis thaliana

2014-01-18T12:01:43Z

MatjaZalar: /* METODE IN REZULTATI */

=='''UVOD'''==
Cvetenje (angl. ‘flowering’) je kompleksno uravnan rastlinski proces, ki se prične kot odgovor na različne okoljske in endogene dejavnike. Znanstveniki so z genetskimi študijami določili štiri glavne skupine, ki kontrolirajo proces cvetenja: vernalizacijska pot, fotoperiodična pot, avtonomna in giberelinska pot. Vernalizacija je indukcija cvetenja rastline po daljšem času mraza. Fotoperiodična pot se nanaša na regulacijo cvetenja glede na dolžino in kakovost prejete svetlobe. Za normalen fenotip cvetenja po giberelinski poti je potrebna prisotnost giberelinske kisline. Avtonomna pot pa regulira cvetenje preko endogenih regulatorjev, ki so neodvisni od fotoperiodne in giberelinske poti. Znanstveniki so identificirali veliko genov, ki regulirajo čas cvetenja (angl. ‘flowering time’) v ''Arabidopsis thailana'' in se medsebojno povezujejo. Mutacije v tovrstnih genih se fenotipsko odražajo v zakasnitvi cvetenja. V mnogih vrstah rastlin je za to potreben prehod iz juvenilne v odraslo fazo, ki pa je reguliran preko avtonomne poti.
Namen raziskave je bila določitev pomembnosti dveh transkripcijskih faktorjev (TF), atVOZ1 in atVOZ2 pri razvoju in cvetenju rastline ''Arabidopsis thailana''.
=='''METODE IN REZULTATI'''==
V raziskavi so uporabili rastlino A. thaliana in mutantne linije znotraj enakega ekotipa. Poskuse so delali na dveh enojnih mutantah (atVOZ1-1 in atVOZ2-1), na dvojni mutanti (atVOZ1-1 atVOZ2-1) in na dvojni mutanti, ki je bila komplementirana z atVOZ2 (atVOZ2; atVOZ1-1 atVOZ2-1) ter jih primerjali z divjim tipom. . V začetnih eksperimentih so opazovali fenotip vseh mutantov ter jih primerjali z divjim sevom. Merili so čas cvetenja, ki je predstavljal čas od dneva saditve do prvega cveta in število rozetnih lističev. Čas cvetenja dvojnih mutant je daljši kot pri divjem sevu, medtem ko v primeru enojnih mutant velikega odstopanja od divjega seva ni bilo, a je bilo še vedno statistično signifikantno. Število rozetnih lističev je bilo pri dvojnih mutantah v DD več kot pri divjem sevu.
Naslednja stopnja poskusa je bila potrditev rezultatov, pridobljenih z opazovanjem fenotipa, na molekularni ravni, z različnimi molekularnimi tehnikami. Z metodo RT-PCR so analizirali ekspresijo genov v vseh mutantah in jih primerjali z divjim tipom, ki so bili že iz prejšnjih raziskav znani, da regulirajo proces cvetenja. Flowering locus C (FLC) je gen, ki kodira protein s funkcijo represije cvetenja in le-ta je bil nadizražen v dvojnih mutantah in prav tako velja za preostale preiskovane regulatorje FLC-ja. Gena FD in FT kodirata proteina, ki promovirata cvetenje. V dvojnih mutantah je bil FD podizražen, medtem ko za FT ni bilo opaznih sprememb. S tem eksperimentom so potrdili značilni fenotip zaostalega cvetenja dvojnih mutant, saj so geni, ki so vključeni v represijo tega procesa nadizraženi, aktivatorji pa so podizraženi.
Znanstvenike je zanimala odzivnost dvojnih mutant po tem, ko so bila semena dvanajst tednov izpostavljena vernalizacijskim pogojem. Opazovali so fenotip, torej cvetni čas in število rozetnih listov ter izražanje gena FLC divjega tipa in dvojnih mutant pri rastlinah, ki so bile izpostavljene pogojem vernalizacije in tistim, ki niso bile. Opazili so, da se je v dvojnih mutantah nivo transkripta gena FLC znižal in rastline so začele cveteti mnogo prej kot dvojne mutante v nevernatalizacijskih pogojih.
Znanstveniki so želeli preučiti interakcije atVOZ proteinov s specifičnimi promotorji genov, ki so bili že predmet raziskav tekom te študije. Ustreznega zaporedja, znanega tudi kot vezavo mesto za VOZ, niso našli v nobeni promotorski regiji, niti gena FLC, niti njegovih regulatorjev kar nakazuje, da ni direktne povezave med TF-jema in prej opisanimi geni. Povezava pa je lahko posredna, zato so iskanje vezavega mesta za VOZ razširili na 1000 bp navzgor od promotorskih zaporedij. Našli so ustrezno vezavno mesto na promotorju gena Modifier of SNC1,3/Supressor of auxin resistance 3 (MOS3/SAR3), ki kodira jedrni membranski protein. V tem eksperimentu so znanstveniki želeli preučiti nivo izražanja MOS3/SAR3 v dvojnih mutantah ter preučiti interakcije atVOZ proteinov z DNA zaporedjem gena MOS3/SAR3 s pomočjo testa zamika elektroforezne mobilnosti (EMSA). Ugotovili so, da je gen MOS3/SAR3 nadizražen in da je vezava atVOZ2 na promotorsko regijo MOS3/SAR3 odvisna od koncentracije proteina. Poleg tega pa so tudi dokazali, da se atVOZ2 na vezavno mesto za VOZ veže s Zn2+ vezavno domeno in tako regulira izražanje gena MOS3/SAR3. To so prav tako določili z metodo EMSA, le da so opazovali vezavo mutiranega atVOZ2 proteina, z mutacijami narejenimi v vezavni domeni, na promotorsko regijo ter vezavo atVOZ2 na vezavnem mesto za VOZ, znotraj katerega je bila narejena mutacija. Zaradi težav pri izražanju atVOZ1 proteina tega zanj niso mogli dokazati, ampak so do podobnih rezultatov prišli z metodo kromatinske imunoprecipitacije (ChIP).

=='''ZAKLJUČEK'''==
Raziskava potrjuje pomembno vlogo transkripcijskih faktorjev atVOZ1 in atVOZ2 v regulaciji prehoda iz vegetativne rasti v cvetenje. Z različnimi eksperimenti, ki si sledijo v logičnem zaporedju so dokazali, da imajo rastline (''atVOZ1-1 atVOZ2-1'') daljši čas cvetenja, kadar le-tega primerjamo z divjim sevom. Dvojni mutanti so občutljivi na vernalizacijo kar se vidi v fenotipu, ki je podoben divjemu sevu in v znižanju izražanja gena FLC. Prav tako pa so dokazali, da transkripcijska faktorja posredno vplivata na gene, ki sodelujejo pri prehodu iz vegetativne rasti v cvetenje, in zagotovo ima del vloge pri tem gen MOS3/SAR3, na katerega transkripcijska faktorja delujeta neposredno.
=='''ČLANEK'''==
CELESNIK, H., ALI, G. S., ROBISON, F. M. & REDDY, A. S. 2013. Arabidopsis thaliana VOZ (Vascular plant One-Zinc finger) transcription factors are required for proper regulation of flowering time. Biol Open, 2, 424-31.

Regulation of flowering time in Arabidopsis thaliana

2014-01-18T11:46:06Z

MatjaZalar: New page: =='''UVOD'''== Cvetenje (angl. ‘flowering’) je kompleksno uravnan rastlinski proces, ki se prične kot odgovor na različne okoljske in endogene dejavnike. Znanstveniki so z genetskimi...

Seminarji TehDNA

2014-01-18T11:12:20Z

MatjaZalar:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

'''SEMINARJI SO OB TORKIH (ob 14h na dekanatu, soba D30, 1. nadstropje levo).'''

Seznam tem za seminarje:

# Mutageneza (16.10.), 3 seminarji: 1. Urban Bezeljak (CRISPR/Cas9) 2. Uroš Stupar (ZFN nukleaze) 3. Helena Vajović (tarčna mutageneza)
# Izražanje na površini (23.10.), 3 seminarji: 1. Mitja Crček (Ribosome display), 2. Klara Tereza Novoselc (Phage display) 3. Živa Marsetič (Predstavitev na površini bakterij)
# Dvohibridni sistemi (30.10.), 3 seminarji: 1. Katja Kovačič (BiFC) 2. Barbara Žužek (YTH s knjižnico celic HeLa) 3. Bernarda Majc (YTH)
# Mutageneza/genetika (6.11.), skupaj 3 seminarji: 1. Valter Bergant (scFv phage display), 2. Ana Kapraljević (gene silencing shRNA), 3. Tjaša Blatnik (gene overexpression)
# GSO v agronomiji (12.11.), 3 seminarji: 1. Niki Bursič (izboljšanje tolerance na mraz in slanost), 2. Petra Malavašič (biofortifikacija riža), 3. Jernej Mustar (rizobakterijska simbioza)
# Transgenske živali (26.11.), 3 seminarji: 1. Andrea Grof (transgenske kokoši), 2. Eva Lucija Kozak (HMC), 3. Špela Pohleven (transgene podgane)
# Izvorne celice (3.12.), 4 seminarji: 1. Sara Primec, 2. Alja Zottel, 3. Tjaša Goričan (SCNT), 4. Rok Štemberger
# DNA-diagnostika (10.12.), 4 seminarji: 1. Tina Gregorič , 2. Eva Knapič, 3. Veronika Jarc, 4. Jana Verbančič
# Forenzika, arheologija, sistematika (17.12.), 3 seminarji: 1. Matja Zalar, 2. Andreja Bratovš, 3. Maja Remškar
# Mikromreže, genomike (7.1.), 3 seminarji: 1. Andrej Vrankar (microarray-gene chips), 2. Filip Kolenc 3. Nastja Štemberger
# Gensko zdravljenje (14.1.), 3 seminarji: 1. Ana Dolinar 2. Staša Komljenovič, 3. Katarina Uršič

'''POVZETEK ZA SEMINAR 21.1.2014''' '''Dolžina predstavitve: 13 min'''

# Tamara Marić ([[Regulation of flowering time in ''Arabidopsis thaliana'']])

'''POVZETKI ZA SEMINARJE 14.1.2014''' '''Dolžina predstavitve: 13 min'''

# Ana Dolinar ([[Retinal Gene Therapy]]) Vprašanja: Tamara Marić, Primec Sara, Jarc Veronika
# Staša Komljenovič ([[Nanoparticle Platform for Delivery of siRNA and Cisplatin]]) Vprašanja: Vrankar Andrej, Grof Andrea, Štemberger Nastja
# Katarina Uršič ([[Combined Cancer Gene Therapy]]) Vprašanja: Kozak Eva Lucija, Vajović Helena

'''POVZETKI ZA SEMINARJE 7.1.2014''' '''Dolžina predstavitve: 13 min'''

# Andrej Vrankar ([[microarray-gene chips]]) Vprašanja: Mustar Jernej, Žužek Barbara
# Filip Kolenc ([[integration of multiple omics datasets]]) Vprašanja: Blatnik Tjaša, Majc Bernarda
# Nastja Štemberger ([[metabolomics]]) Vprašanja: Kapraljević Ana, Zottel Alja

'''POVZETKI ZA SEMINARJE 17.12.2013''' '''Dolžina predstavitve: 13 min'''

'''Študente naprošam, da si preberejo članek Maje Remškar, ker bomo v torek za ta članek skupaj sestavili recenzijo. Izbrala sem ga, ker je zelo kratek in enostaven, tako da vam ne bo vzel preveč časa. Hvala, Helena Čelešnik'''

# Matja Zalar ([[Arheologija, Whole-Genome Capture Method]]) Vprašanja: Goričan Tjaša, Grof Andrea
# Andreja Bratovš ([[Sistematika, Human Gut Microbiome]]) Vprašanja: Kozak Eva Lucija, Pohleven Špela
# Maja Remškar ([[Forenzika, Determining the Body Fluid Origin]]) Vprašanja: Štemberger Rok, Verbančič Jana

'''POVZETKI ZA SEMINARJE 10.12.2013:''' '''dolžina predstavitve: 13 min, čas za vprašanja: 9 min na seminar.

PROSIM, DA SE STRIKTNO DRŽITE DOLŽINE PREDSTAVITEV, KER IMAMO TA TEDEN 4 SEMINARJE
# Tina Gregorič ([[Comparative genomic hybridization (CGH) arrays]]) Vprašanja: Crček Mitja, Majc Bernarda
# Eva Knapič ([[Automated Multiplexing Quantum Dots in Situ Hybridization Assay ]]) Vprašanja: Stupar Uroš, Malavašič Petra
# Veronika Jarc ([[identification and mapping of clone-specific chromosomal abnormalities ]]) Vprašanja: Marsetič Żiva, Bursić Niki
# Jana Verbančič ([[multiplex oligonucleotide ligation assay]]) Vprašanja: Bezeljak Urban, Żużek Barbara

'''POVZETKI ZA SEMINARJE 3.12.2013:''' '''dolžina predstavitve: 13 min, čas za vprašanja: 9 min na seminar.

PROSIM, DA SE STRIKTNO DRŽITE DOLŽINE PREDSTAVITEV, KER IMAMO TA TEDEN 4 SEMINARJE
# Sara Primec([[Stem Cell Educator therapy]]) Vprašanja: Bergant Valter, Novoselc Klara Tereza
# Alja Zottel ([[hematopoietic stem/progenitor cells (HSPC) modification]]) Vprašanja: Dolinar Ana, Kapraljević Ana
# Tjaša Goričan ([[Bone Marrow Mesenchymal Stem Cells & SCNT]]) Vprašanja: Vrankar Andrej, Uršič Katarina
# Rok Štemberger ([[neural stem cells]]) Vprašanja: Komljenović Staša, Blatnik Tjaša

'''POVZETKI ZA SEMINARJE 26.11.2013 (ob 14h, soba D30)''' '''Dolžina predstavitve: 13 min'''
# Andrea Grof ([[transgenske kokoši]]) Vprašanja: Zottel Alja, Vajović Helena
# Eva Lucija Kozak ([[HMC]]) Vprašanja: Štemberger Nastja, Knapič Eva
# Špela Pohleven ([[transgene podgane]]) Vprašanja: Bratovš Andreja, Kovačič Katja

'''POVZETKI ZA SEMINARJE 12.11.2013 (ob 14h na dekanatu - D30)''' '''Dolžina predstavitve: 13 min'''
# Niki Bursič ([[izboljšanje tolerance na mraz in slanost]]) (Vsak svoje) vprašanje postavita Kolenc Filip in Primec Sara
# Petra Malavašič ([[biofortifikacija riža]]) Vprašanje postavita Zalar Matja in Gregorič Tina
# Jernej Mustar ([[rizobakterijska simbioza]]) Vprašanje postavita Jarc Veronika in Remškar Maja

'''POVZETKI ZA SEMINARJE 6.11.2013'''
# Valter Bergant ([[scFv phage display]])
# Ana Kapraljević ([[gene silencing shRNA]])
# Tjaša Blatnik ([[gene overexpression]])

'''POVZETKI ZA SEMINARJE 30.10.2013'''
# Katja Kovačič ([[BiFC]])
# Barbara Žužek ([[YTH s knjižnico celic HeLa]])
# Bernarda Majc ([[YTH]])

'''POVZETKI ZA SEMINARJE 23.10.2013'''
# Mitja Crček ([[Predstavitev na ribosomih]])
# Klara Tereza Novoselc ([[Phage display]])
# Živa Marsetič ([[Predstavitev na površini bakterij]])

'''POVZETKI ZA SEMINARJE 14.10.2013'''
# Urban Bezeljak ([[CRISPR/Cas9]])
# Uroš Stupar ([[ZFN nukleaze]])
# Helena Vajović ([[tarčna mutageneza]])

Seminarji TehDNA

2014-01-18T11:10:16Z

MatjaZalar:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

'''SEMINARJI SO OB TORKIH (ob 14h na dekanatu, soba D30, 1. nadstropje levo).'''

Seznam tem za seminarje:

# Mutageneza (16.10.), 3 seminarji: 1. Urban Bezeljak (CRISPR/Cas9) 2. Uroš Stupar (ZFN nukleaze) 3. Helena Vajović (tarčna mutageneza)
# Izražanje na površini (23.10.), 3 seminarji: 1. Mitja Crček (Ribosome display), 2. Klara Tereza Novoselc (Phage display) 3. Živa Marsetič (Predstavitev na površini bakterij)
# Dvohibridni sistemi (30.10.), 3 seminarji: 1. Katja Kovačič (BiFC) 2. Barbara Žužek (YTH s knjižnico celic HeLa) 3. Bernarda Majc (YTH)
# Mutageneza/genetika (6.11.), skupaj 3 seminarji: 1. Valter Bergant (scFv phage display), 2. Ana Kapraljević (gene silencing shRNA), 3. Tjaša Blatnik (gene overexpression)
# GSO v agronomiji (12.11.), 3 seminarji: 1. Niki Bursič (izboljšanje tolerance na mraz in slanost), 2. Petra Malavašič (biofortifikacija riža), 3. Jernej Mustar (rizobakterijska simbioza)
# Transgenske živali (26.11.), 3 seminarji: 1. Andrea Grof (transgenske kokoši), 2. Eva Lucija Kozak (HMC), 3. Špela Pohleven (transgene podgane)
# Izvorne celice (3.12.), 4 seminarji: 1. Sara Primec, 2. Alja Zottel, 3. Tjaša Goričan (SCNT), 4. Rok Štemberger
# DNA-diagnostika (10.12.), 4 seminarji: 1. Tina Gregorič , 2. Eva Knapič, 3. Veronika Jarc, 4. Jana Verbančič
# Forenzika, arheologija, sistematika (17.12.), 3 seminarji: 1. Matja Zalar, 2. Andreja Bratovš, 3. Maja Remškar
# Mikromreže, genomike (7.1.), 3 seminarji: 1. Andrej Vrankar (microarray-gene chips), 2. Filip Kolenc 3. Nastja Štemberger
# Gensko zdravljenje (14.1.), 3 seminarji: 1. Ana Dolinar 2. Staša Komljenovič, 3. Katarina Uršič

'''POVZETKI ZA SEMINARJE 21.1.2014''' '''Dolžina predstavitve: 13 min'''

# Tamara Marić ([[Regulation of flowering time in ''Arabidopsis thaliana'']])

'''POVZETKI ZA SEMINARJE 14.1.2014''' '''Dolžina predstavitve: 13 min'''

# Ana Dolinar ([[Retinal Gene Therapy]]) Vprašanja: Tamara Marić, Primec Sara, Jarc Veronika
# Staša Komljenovič ([[Nanoparticle Platform for Delivery of siRNA and Cisplatin]]) Vprašanja: Vrankar Andrej, Grof Andrea, Štemberger Nastja
# Katarina Uršič ([[Combined Cancer Gene Therapy]]) Vprašanja: Kozak Eva Lucija, Vajović Helena

'''POVZETKI ZA SEMINARJE 7.1.2014''' '''Dolžina predstavitve: 13 min'''

# Andrej Vrankar ([[microarray-gene chips]]) Vprašanja: Mustar Jernej, Žužek Barbara
# Filip Kolenc ([[integration of multiple omics datasets]]) Vprašanja: Blatnik Tjaša, Majc Bernarda
# Nastja Štemberger ([[metabolomics]]) Vprašanja: Kapraljević Ana, Zottel Alja

'''POVZETKI ZA SEMINARJE 17.12.2013''' '''Dolžina predstavitve: 13 min'''

'''Študente naprošam, da si preberejo članek Maje Remškar, ker bomo v torek za ta članek skupaj sestavili recenzijo. Izbrala sem ga, ker je zelo kratek in enostaven, tako da vam ne bo vzel preveč časa. Hvala, Helena Čelešnik'''

# Matja Zalar ([[Arheologija, Whole-Genome Capture Method]]) Vprašanja: Goričan Tjaša, Grof Andrea
# Andreja Bratovš ([[Sistematika, Human Gut Microbiome]]) Vprašanja: Kozak Eva Lucija, Pohleven Špela
# Maja Remškar ([[Forenzika, Determining the Body Fluid Origin]]) Vprašanja: Štemberger Rok, Verbančič Jana

'''POVZETKI ZA SEMINARJE 10.12.2013:''' '''dolžina predstavitve: 13 min, čas za vprašanja: 9 min na seminar.

PROSIM, DA SE STRIKTNO DRŽITE DOLŽINE PREDSTAVITEV, KER IMAMO TA TEDEN 4 SEMINARJE
# Tina Gregorič ([[Comparative genomic hybridization (CGH) arrays]]) Vprašanja: Crček Mitja, Majc Bernarda
# Eva Knapič ([[Automated Multiplexing Quantum Dots in Situ Hybridization Assay ]]) Vprašanja: Stupar Uroš, Malavašič Petra
# Veronika Jarc ([[identification and mapping of clone-specific chromosomal abnormalities ]]) Vprašanja: Marsetič Żiva, Bursić Niki
# Jana Verbančič ([[multiplex oligonucleotide ligation assay]]) Vprašanja: Bezeljak Urban, Żużek Barbara

'''POVZETKI ZA SEMINARJE 3.12.2013:''' '''dolžina predstavitve: 13 min, čas za vprašanja: 9 min na seminar.

PROSIM, DA SE STRIKTNO DRŽITE DOLŽINE PREDSTAVITEV, KER IMAMO TA TEDEN 4 SEMINARJE
# Sara Primec([[Stem Cell Educator therapy]]) Vprašanja: Bergant Valter, Novoselc Klara Tereza
# Alja Zottel ([[hematopoietic stem/progenitor cells (HSPC) modification]]) Vprašanja: Dolinar Ana, Kapraljević Ana
# Tjaša Goričan ([[Bone Marrow Mesenchymal Stem Cells & SCNT]]) Vprašanja: Vrankar Andrej, Uršič Katarina
# Rok Štemberger ([[neural stem cells]]) Vprašanja: Komljenović Staša, Blatnik Tjaša

'''POVZETKI ZA SEMINARJE 26.11.2013 (ob 14h, soba D30)''' '''Dolžina predstavitve: 13 min'''
# Andrea Grof ([[transgenske kokoši]]) Vprašanja: Zottel Alja, Vajović Helena
# Eva Lucija Kozak ([[HMC]]) Vprašanja: Štemberger Nastja, Knapič Eva
# Špela Pohleven ([[transgene podgane]]) Vprašanja: Bratovš Andreja, Kovačič Katja

'''POVZETKI ZA SEMINARJE 12.11.2013 (ob 14h na dekanatu - D30)''' '''Dolžina predstavitve: 13 min'''
# Niki Bursič ([[izboljšanje tolerance na mraz in slanost]]) (Vsak svoje) vprašanje postavita Kolenc Filip in Primec Sara
# Petra Malavašič ([[biofortifikacija riža]]) Vprašanje postavita Zalar Matja in Gregorič Tina
# Jernej Mustar ([[rizobakterijska simbioza]]) Vprašanje postavita Jarc Veronika in Remškar Maja

'''POVZETKI ZA SEMINARJE 6.11.2013'''
# Valter Bergant ([[scFv phage display]])
# Ana Kapraljević ([[gene silencing shRNA]])
# Tjaša Blatnik ([[gene overexpression]])

'''POVZETKI ZA SEMINARJE 30.10.2013'''
# Katja Kovačič ([[BiFC]])
# Barbara Žužek ([[YTH s knjižnico celic HeLa]])
# Bernarda Majc ([[YTH]])

'''POVZETKI ZA SEMINARJE 23.10.2013'''
# Mitja Crček ([[Predstavitev na ribosomih]])
# Klara Tereza Novoselc ([[Phage display]])
# Živa Marsetič ([[Predstavitev na površini bakterij]])

'''POVZETKI ZA SEMINARJE 14.10.2013'''
# Urban Bezeljak ([[CRISPR/Cas9]])
# Uroš Stupar ([[ZFN nukleaze]])
# Helena Vajović ([[tarčna mutageneza]])

Arheologija, Whole-Genome Capture Method

2013-12-12T22:42:23Z

MatjaZalar:

'''UVOD'''
Poznavanje genoma arheološko zanimivih primerkov je pomembno predvsem pri analizah v populacijski genetiki, na podlagi katerih se raziskuje preseljevanje narodov, ugotavlja zgodovinske povezave med posameznimi območji, ipd. Za natančno določitev genoma je potreben dovolj čist vzorec DNA. Glavni problem, s katerim se srečujejo znanstveniki, je propadanje DNA s časom. Tako se je izkazalo, da je v vzorcih teh starodavnih osebkov običajno manj kot 1% endogene DNA, vse ostalo pa predstavlja DNA iz okolja. Poleg tega je DNA razgrajena v majhne fragmente, kar dodatno oteži določevanje nukleotidnega zaporedja.
=='''METODE'''==
V predstavljeni študiji so razvili metodo zajema celotnega genoma v raztopini (anj: whole-genome in-solution capture) ali na kratko WISC, s katero nespecifično povečajo vsebnost endogene DNA v aDNA knjižnici, in njeno učinkovitost preverili na dvanajstih vzorcih, pridobljenih iz kosti, zob ali las ljudi, ki so živeli v železni oziroma bakreni dobi na območju Bolgarije, Peruja in Danske.
DNA so iz vzorcev tkiva ekstrahirali po metodi, ki temelji na vezavi DNA na kolone s silicijevim dioksidom. Očiščeni DNA so popravili konce in dodali dA-rep, nato pa na njih vezali specifične Illumina adapterje. Nato so izvedli sekvenciranje knjižnic pred zajetjem endogene DNA.
Za vabo, s katero so iz vzorca aDNA pridobili endogeno DNA, so pripravili biotinilirano RNA knjižnico človeškega genoma, s fragmenti dolgimi 200 – 300 bp. Na konce so jim pripeli T7 adapterje. V hibridizacijsko mešanico so dodali tudi oligonukleotide, ki so se vezali na adapterje ter preprečili nespecifične vezave preko adapterjev. Po hibridizaciji so z magnetnimi delci, na katerih je bil vezan streptavidin, iz raztopine ločili nevezane RNA-vabe in eksogeno DNA ter RNA vabe, ki so se hibridizirale na aDNA. Nato pa izvedli drugo sekvenciranje, tokrat knjižnic po zajetju endogene DNA.
=='''REZULTATI'''==
Analiza rezultatov sekvenciranja je pokazala, da se po uporabi metode WISC število odčitkov, ki so pripadali človeškemu genomu, poveča za 6-159-krat. V obeh knjižnicah je bila sestava eksogene DNA podobna, le da jo je bilo v knjižnicah po zajetju endogene DNA v vzorcu količinsko prisotne manj. V petih od dvanajstih knjižnic, pridobljenih po zajetju endogene DNA, so že z relativno kratkim sekvenciranjem dosegli zadostno pokrivanje mtDNA za okvirno določitev skupine haplotipa.
Za vsako knjižnico posebej so določili število unikatnih SNP-jev, ki so jih uporabili za PCA analizo. PCA analiza je statistična metoda, s katero lahko najdemo linearne kombinacije markerjev, ki so značilni za različne genetske skupine. V knjižnicah, narejenih po zajetju endogene DNA, se je povečalo število unikatnih SNP-jev, kar je bistveno izboljšalo tudi statistično zanesljivost PCA analize. Rezultati so bili pričakovani – evropski vzorci so se uvrstili v območje značilno za evropejce, perujski vzorci pa so bili indijanskega porekla.
Ugotovili so, da število unikatnih fragmentov v knjižnicah pred zajetjem endogene DNA linearno narašča s povečevanjem števila odčitkov, medtem ko v knjižnicah po zajetju število doseže plato že pri 4.000.000 očitkov, nato pa ne narašča več. Podoben trend se je pojavil tudi pri šestih kasneje testiranih knjižnicah.Za aDNA je značilna velika fragmentacija in povečana vsebnost G-C parov v primerjavi s A-T pari na konceh fragmentov. Pri primerjavi knjižnic pred in po zajetju endogene DNA se je izkazalo, da so vzorcu poškodb pri obeh skupinah podobni in značilni za aDNA.
=='''ZAKLJUČEK'''==
V predstavljeni raziskavi so razvili prvo metodo zajema celotnega genoma v raztopini ali na kratko WISC, s katero so nespecifično povečali vsebnost endogene DNA v aDNA knjižnici. Glavni problem te metode pa je premajhna specifičnost, zaradi katere knjižnice aDNA vsebujejo premalo endogene DNA za pokritje celotnega genoma. Uspešnost zajema genomskih fragmetov bi lahko izboljšali z optimizacijo ekstrakcije aDNA iz vzorcev tkiva in postopka priprave aDNA knjižnic.
Kljub nekaterim pomankljivostim je metoda perspektiven pristop k enostavnejšem sekvenciranju aDNA
=='''ČLANEK'''==
Carpenter et al., Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA
Sequencing Libraries, The American Journal of Human Genetics (2013), http://dx.doi.org/10.1016/j.ajhg.2013.10.002

Arheologija, Whole-Genome Capture Method

2013-12-12T22:41:17Z

MatjaZalar: New page: '''UVOD''' Poznavanje genoma arheološko zanimivih primerkov je pomembno predvsem pri analizah v populacijski genetiki, na podlagi katerih se raziskuje preseljevanje narodov, ugotavlja zg...

'''UVOD'''
Poznavanje genoma arheološko zanimivih primerkov je pomembno predvsem pri analizah v populacijski genetiki, na podlagi katerih se raziskuje preseljevanje narodov, ugotavlja zgodovinske povezave med posameznimi območji, ipd. Za natančno določitev genoma je potreben dovolj čist vzorec DNA. Glavni problem, s katerim se srečujejo znanstveniki, je propadanje DNA s časom. Tako se je izkazalo, da je v vzorcih teh starodavnih osebkov običajno manj kot 1% endogene DNA, vse ostalo pa predstavlja DNA iz okolja. Poleg tega je DNA razgrajena v majhne fragmente, kar dodatno oteži določevanje zaporedja.
=='''METODE'''==
V predstavljeni študiji so razvili metodo zajema celotnega genoma v raztopini (anj: whole-genome in-solution capture) ali na kratko WISC, s katero nespecifično povečajo vsebnost endogene DNA v aDNA knjižnici, in njeno učinkovitost preverili na dvanajstih vzorcih, pridobljenih iz kosti, zob ali las ljudi, ki so živeli v železni oziroma bakreni dobi na območju Bolgarije, Peruja in Danske.
DNA so iz vzorcev tkiva ekstrahirali po metodi, ki temelji na vezavi DNA na kolone s silicijevim dioksidom. Očiščeni DNA so popravili konce in dodali dA-rep, nato pa na njih vezali specifične Illumina adapterje. Nato so izvedli sekvenciranje knjižnic pred zajetjem endogene DNA.
Za vabo, s katero so iz vzorca aDNA pridobili endogeno DNA, so pripravili biotinilirano RNA knjižnico človeškega genoma, s fragmenti dolgimi 200 – 300 bp. Na konce so jim pripeli T7 adapterje. V hibridizacijsko mešanico so dodali tudi oligonukleotide, ki so se vezali na adapterje ter preprečili nespecifične vezave preko adapterjev. Po hibridizaciji so z magnetnimi delci, na katerih je bil vezan streptavidin, iz raztopine ločili nevezane RNA-vabe in eksogeno DNA ter RNA vabe, ki so se hibridizirale na aDNA. Nato pa izvedli drugo sekvenciranje, tokrat knjižnic po zajetju endogene DNA.
=='''REZULTATI'''==
Analiza rezultatov sekvenciranja je pokazala, da se po uporabi metode WISC število odčitkov, ki so pripadali človeškemu genomu, poveča za 6-159-krat. V obeh knjižnicah je bila sestava eksogene DNA podobna, le da jo je bilo v knjižnicah po zajetju endogene DNA v vzorcu količinsko prisotne manj. V petih od dvanajstih knjižnic, pridobljenih po zajetju endogene DNA, so že z relativno kratkim sekvenciranjem dosegli zadostno pokrivanje mtDNA za okvirno določitev skupine haplotipa.
Za vsako knjižnico posebej so določili število unikatnih SNP-jev, ki so jih uporabili za PCA analizo. PCA analiza je statistična metoda, s katero lahko najdemo linearne kombinacije markerjev, ki so značilni za različne genetske skupine. V knjižnicah, narejenih po zajetju endogene DNA, se je povečalo število unikatnih SNP-jev, kar je bistveno izboljšalo tudi statistično zanesljivost PCA analize. Rezultati so bili pričakovani – evropski vzorci so se uvrstili v območje značilno za evropejce, perujski vzorci pa so bili indijanskega porekla.
Ugotovili so, da število unikatnih fragmentov v knjižnicah pred zajetjem endogene DNA linearno narašča s povečevanjem števila odčitkov, medtem ko v knjižnicah po zajetju število doseže plato že pri 4.000.000 očitkov, nato pa ne narašča več. Podoben trend se je pojavil tudi pri šestih kasneje testiranih knjižnicah.Za aDNA je značilna velika fragmentacija in povečana vsebnost G-C parov v primerjavi s A-T pari na konceh fragmentov. Pri primerjavi knjižnic pred in po zajetju endogene DNA se je izkazalo, da so vzorcu poškodb pri obeh skupinah podobni in značilni za aDNA.
=='''ZAKLJUČEK'''==
V predstavljeni raziskavi so razvili prvo metodo zajema celotnega genoma v raztopini ali na kratko WISC, s katero so nespecifično povečali vsebnost endogene DNA v aDNA knjižnici. Glavni problem te metode pa je premajhna specifičnost, zaradi katere knjižnice aDNA vsebujejo premalo endogene DNA za pokritje celotnega genoma. Uspešnost zajema genomskih fragmetov bi lahko izboljšali z optimizacijo ekstrakcije aDNA iz vzorcev tkiva in postopka priprave aDNA knjižnic.
Kljub nekaterim pomankljivostim je metoda perspektiven pristop k enostavnejšem sekvenciranju aDNA
=='''ČLANEK'''==
Carpenter et al., Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA
Sequencing Libraries, The American Journal of Human Genetics (2013), http://dx.doi.org/10.1016/j.ajhg.2013.10.002

Seminarji TehDNA

2013-10-08T15:06:02Z

MatjaZalar:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

Seznam tem za seminarje:

# Mutageneza (16.10.), 3 seminarji:
# Izražanje na površini (23.10.), 3 seminarji:
# Dvohibridni sistemi (30.10.), 3 seminarji: 1.Rok Štemberger
# Mikromrežne tehnologije (6.11.), 3 seminarji:
# GSO v agronomiji (13.11.), 3 seminarji: 1. Niki Bursič, 2. Petra Malavašič, 3. Jernej Mustar
# Transgenske živali (27.11.) Andrea Grof, Eva Lucija Kozak, Špela Pohleven
# Izvorne celice (4.12.) Sara Primec, Alja Zottel, Tjaša Goričan
# DNA-diagnostika (11.12.) Tina Gregorič , Eva Knapič, Veronika Jarc
# Forenzika, arheologija, sistematika (18.12.) Matja Zalar
# Mikromreže, genomike (8.1.)
# Gensko zdravljenje s. lat. (15.1.) Ana Dolinar

Možni pristopi k zdravljenju raka z RNAi

2012-04-10T18:26:02Z

MatjaZalar:

==Uvod==
Z imenom rak označujemo skupino bolezni, katerih skupna značilnost je nenadzorovana proliferacija in sposobnost napadanja drugih tkiv. Danes najpogostejše oblike zdravljenja so operativni posegi, zdravljenje z zdravili, ki pobijajo rakaste celice ali zavirajo njihovo rast, radioterapija in imunoterapija. Naštete metode so precej nespecifične in invazivne zato poleg rakavih celic močno prizadanejo tudi zdravo tkivo, kar se odraža v hudih stranskih učinkih. Delovanje RNAi pa je usmerjeno na en ali na skupek sorodnih genov, zato je učinek lokaliziran le na mutirane celice.

==Vpliv RNAi v tumorskih celicah==
Princip delovanja RNAi je pri tumorskih celicah popolnoma enak kot pri normalnih celicah.
Ugotovljeno pa je bilo, da je izražanje miRNA pri rakavih celicah drugačno kot pri normalnih celicah, pri čemer se poveča predvsem ekspresija onkogenske miRNA. Na podlagi izražene miRNA je možno ugotoviti tako vrsto raka kot tudi stadij njegovega razvoja. Z induciranim spreminjanjem ekspresije miRNA pa je možno doseči tudi spremembe v fenotipu raka. Prav ti dve značilnosti sta osnova za razvoj zdravljenja z RNAi.

===Potencialne tarče zdravljenja===
S pomočjo študij mutacij, ki se pojavljajo v malignih tumorjih, so ugotovili, da so v rakavih celicah najpogostejše spremembe genov, ki zapisujejo za:
*proteine,ki sodelujejo pri regulaciji celičnega cikla,kot sta P53 in PRb,
*tirozin kinaze,
*pro- in antiapoptotske proteine,
*proteine povezane s celičnim staranjem,
*proteine, ki celicam omogočijo pritrditev na podlago in medsebojno povezovanje,
*proteine, ki vplivajo na razgradnjo in stabilnost drugih proteinov.

Poleg zgoraj naštetih so kot tarče za zdravljenje z RNAi uporabni tudi nekateri transkripcijski faktorji (npr. VEGF), ki sodelujejo pri ožiljenju, rasti ali razširjanju tumorjev po telesu.
S pomočjo vnosa sintetične miRNA ali siRNA je možno raziskovanje celičnih procesov, ki so specifični ali okvarjeni v rakavih celicah. Kot primer naj navedem, da so s pomočjo RNAi vektorjev in sistematičnega raziskovanja ter primerjave med izražanjem genov v rakavih in normalinih celicah, identificirali deubikvitinacijske encime in gene, vpletene v proces s TRAIL inducirane apoptoze. Poleg tega lahko s selektivnim sistematičnim utiševanjem posameznih genov raziskujemo vlogo in vpliv posamičnih proteinov na procese, kot so apoptoza in proliferacija, ki so spremenjeni pri praktično vseh vrstah rakov. S takšnim načinom raziskovanja so določili tudi zgoraj naštete tarče zdravljenja z RNAi.

===Razvoj terapevtikov===
Manipulacija izražanja genov lahko poteka z miRNA, s katero hkrati vplivamo na več genov, ki pa so pogosto medsebojno povezani v smislu skupnega vpliva na določen celični proces, ali s siRNA, ki je specifična za en sam gen.

Ena veja razvoja RNAi terapevtikov želi ustvariti zdravila, ki temeljijo na utišanju antiapoptotskih genov in genov udeleženih pri proliferaciji, s čimer bi rast tumorjev vsaj ustavili, oziroma rakave celice uničili. Druga veja pa razvija terapevtike, ki bi s svojim delovanjem preprečili izražanje proteinov, ki povzročajo rezistenco tumorskih celic na kemoterapevtike. S tem bi dobili dobra podporna zdravila, ki bi izboljšala uspešnost kemoterapije. Pri rakih, za katere je značilna okvara Dicerja, pa zaradi drugačnega načina izražanja miRNA, terapije z RNAi niso uspešne.

===Princip delovanja terapevtikov===
Kot smo že omenili, so razlike fenotipov normalnega in rakavega tkiva osnova za razvoj terapevtikov. Do danes so razvili dva principa delovanja zdravil: neposredno in posredno. Pri neposredni metodi uporabljajo za blokiranje ekspresije onkogene miRNA in nadomeščanje tumor supresorske miRNA, ki je v rakavih celicah manj izražena, oligonukleotide in virusne konstrukte.
Pri posredni metodi uporabljajo zdravila, ki ne delujejo direktno na miRNA, ampak preko kompleksov, ki sodelujejo pri zorenju v zrelo miRNA.

*'''Antisense oligonukleotidi''' delujejo kot kompetitivni inhibitorji na onkogeno miRNA. So komplementarni zaporedju željene miRNA in se ob vstopu v celico nanjo vežejo. Skupaj tvorita dupleks, ki se kasneje razgradi. V prvem poskusu so inhibitorno miRNA vbrizgali v ''D. melanogaster'' in koncentracija onkogene miRNA se je znižala. Ker enak poskus pri ''C. elegans'' ni bil uspešen, so znanstveniki s kemijsko modifikacijo stabilizirali inhibitorno miRNA ter tako povečali njeno specifičnost. miRNA je stabilizirana z metilno ali metoksietilno skupino na 2’-O mestu na ribozi. Delovanje antisense oligonukleotidov so Krutzfeldt ''et al.'' pokazali na primeru miR-122, ki je najbolje izražena v jetrih. V mišjo repno veno so vbrizgali inhibitorno miRNA, drugače imenovano antagomir, in po 24 urah so opazili spremembo v koncentraciji miR-122. S tem so pokazali, da je utišanje genov s to metodo specifično, učinkovito in dolgotrajno, kajti učinek utišanja je trajal 23 dni.

*'''miRNA sponges''' so transkripti z vezavnimi mesti za miRNA, katere delovanje želimo v celici preprečiti. V celici tekmujejo za vezavo onkogene miRNA z mRNA. V celici se prepisujejo iz predhodno vstavljenega vektorja. miRNA ima večjo afiniteto do tega transkripta kot do tarčne mRNA. Prvi razlog za to je popolna komplementarnost zaporedja onkogene miRNA in transkripta, drugi pa umetno narejena izboklina na mestu vezave za Ago2 protein, ki omogoči bolj stabilno vezavo med miRNA sponges in onkogeno miRNA v kompleksu RISC. Eksperiment je pokazal, da miRNA sponges v organizmu učinkujejo podobno kot antisense oligonukleotidi.

*'''Obnova ekspresije tumor supresorske miRNA''': Dejstvo, da je v rakavem tkivu nižji nivo tumor supresorske miRNA v primerjavi z normalnim tkivom, izkoristimo tako, da jo dodamo umetno: intravenozno ali preko adenovirusnih vektorjev. Intravenozno vnašamo miRNA mimike. To so dvovijačne, majhne in kemijsko modificirane molekule, ki inducirajo programirano celično smrt in blokirajo proliferacijo. V številnih tipih rakov je inhibirano izražanje miR-15a in družine miR-29. Eksperiment je pokazal, da ob vnosu teh dveh miRNA mimics v jetrne tumorje ali rabdomiosarkome pride do inducirane celične smrti in posledičnega zmanjšanja tumorskega tkiva. Pri drugi metodi pa z adenovirusi transduciramo vektor v celico, s čimer zagotovimo dolgoročno ekspresijo in minimalno toksičnost. Dokazano je bilo, da je ekspresija miR-26 v jetrnih tumorjih zmanjšana. Kota ''et al''. pa so pokazali, da z vnosom adenovirusnega vektorja miR-26 dosežejo zmanjšanje tumorjev in zmanjšano stopnjo proliferacije.
[http://www.nature.com/nrd/journal/v9/n10/images/nrd3179-f4.jpg Shema principov delovanja terapevtikov]

==miRNA kot pomoč pri diagnosticiranju raka==
Razlike v ekspresiji miRNA v rakavih celicah glede na normalne celice lahko uporabimo tudi za diagnosticiranje vrste raka. Lu ''et al''. so v raziskavi pokazali, da je potrebno le približno 200 genov miRNA za uspešno klasifikacijo človeških rakavih obolenj. Za primerjavo naj navedem podatek, da so podobno klasifikacijo poskušali izvesti na podlagi mRNA. Uporabili so 16.000 genov mRNA, ki kodirajo za proteine, vendar jim ni uspelo najti povrzave med rakom z istim izvorom. Nasprotno pa imajo raki iz iste družine (npr. tumorji endotelijskega izvora: rak na debelem črevesu, rak na jetrih, trebušni slinavki) podoben profil izražanja miRNA. Baza podatkov z značilnimi miRNA profili za različne tumorje bi lahko v prihodnosti olajšala diagnozo in tudi zdravljenje raka, saj je s profilom izražanja miRNA poleg vrste raka možno določiti tudi stopnjo diferenciacije tumorskih celic. Ti dve informaciji pa bi bili v veliko pomoč pri odločitvah o vrsti terapije za pacienta.
Ugotovljeno je bilo tudi, da so pri določenih vrstah raka tudi v krvni plazmi prisotne različne miRNA. miRNA bi tako lahko uporabili kot neinvazivni biomarker za zgodnje odkrivanje raka, vendar so potrebne nadaljne raziskave, s katerimi bi določili normalne nivoje miRNA v krvi.

==Ovire pri zdravljenju raka z RNAi==
*Ena od največjih težav, s katerimi se srečujejo raziskovalci pri razvoju RNAi terapevtikov je '''vnos siRNA/shRNA''' v celico. Molekule RNAi lahko vežejo na prenašalce, od katerih so trenutno najbolj perspektivni različni nanodelci.
*Težavo lahko predstavlja tudi '''nespecifičnost RNAi'''. Čeprav je siRNA specifična za določen gen, lahko pride do hibridizacije s transkripti, ki vsebujejo le delno skladno zaporedje. Včasih je dovolj že 6-8 nukleotidov. Metilacija hidroksilne skupine na ribozi poveča afiniteto siRNA do določenega zaporedja.
*Rakave celice imajo pogosto zmanjšano koncentracijo Dicerja in Droshe, kar vpliva na procesiranje prekurzorjev siRNA. Pride lahko tudi do prenasičenja kompleksov RISC, kar vodi v tekmovanje med eksogeno siRNA/miRNA in endogeno miRNA. Posledično se lahko izrazijo geni, ki jih regulira endogena miRNA.
*Treba je tudi upoštevati, da se normalne celice na vstavljeno RNAi odzovejo drugače kot rakave celice. Ponavadi utišanje traja dlje v normalnem tkivu, saj se rakave celice delijo hitreje in pride do razredčenja interferenčnih molekul.
*'''Rezistenca na RNAi''': Pri siRNA lahko zaradi zelo specifične vezave že pri točkovnih mutacijah v tarčnem zaporedju pride do rezistence tumorskih celic na RNAi. Nekatera zaporedja so lahko tudi nedostopna za siRNA zaradi sekundarnih struktur ali vezanih proteinov. Tem dvem težavam se lahko izognemo tako, da si izberemo več zaporedij na tarčni mRNA in več tarčnih mRNA v sami celici. Z utišanjem genov pa ne moremo vplivati na že obstoječe proteine, zato je tudi dolga življenjska doba proteinov oblika rezistence. Rakave celice lahko postanejo odporne na RNAi tudi z mutacijo ali zmanjšanim izražanjem genov za proteine, ki sodelujejo pri procesiranju siRNA/miRNA (Dicer, Drosha, RISC kompleks). Te mehanizme rezistence bo potrebno še natančneje raziskati, da se bo lahko RNAi uporabljala za zdravljenje raka.

==Perspektive==
Večina farmacevtskih podjetij se ukvarja z razvojem zdravil na osnovi siRNA. Vsako zdravilo mora biti potrjeno v petih fazah kliničnega testiranja. Glede na to, da so vsi RNAi terapevtiki zaenkrat v fazi I ali II, v bližnji prihodnosti še ne moremo pričakovati zdravila za rakava obolenja, ki bi delovalo na osnovi RNA interference. Zaenkrat raziskave temeljijo na manipulaciji ene miRNA ali ene družine miRNA. Znanstveniki predvidevajo, da je razvoj raka odvisen od medsebojnega vpliva različnih miRNA, zato bo za učinkovito zdravljenje najverjetneje potrebno razviti zdravila, ki bodo vplivala na celotno mrežo miRNA v obolelih celicah in ne na eno samo. Izboljšave terapevtikov stremijo k minimalni toksičnosti nosilcev in maksimalni specifičnosti zdravila. Vizija razvoja je izdelava terapevtikov za vse vrste rakov, na katere lahko vplivamo z RNAi.

==Viri==
*Garzon R., Marcucci G., Croce C. M. Targeting microRNA in cancer: rationale, strategies and challenges. ''Nature reviews drug discovery'', 2010, vol. 9, str. 775-789.
*Pai SI ''et al''. Prospects of RNAi interference therapy for cancer. ''Gene therapy'', 2006, vol. 13, str. 464-477.
*Petrocca F. in Lieberman J. Promise and challenge of RNA interference - based therapy for cancer. ''Journal of clinical oncology'', 2011, letn. 29, štev. 9, str. 747-754.
*Pecot C. V. et al. RNA interference in the clinic: challenges and future directions. ''Nature reviews'', 2011, vol. 11, str. 59-67.
*Esquela-Kerscher, A. in Slack, F. J. Oncomirs — microRNAs with a role in cancer. ''Nature Reviews Cancer'', 2006, štev. 6, str. 259-269

[[Category:SEM]] [[Category:BMB]]

BIO2 Seminar 2011

2011-11-04T13:36:31Z

MatjaZalar: /* Seznam seminarjev- datumi in seznam recenzentov še niso dokončni! */

= Biokemijski seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsako sredo in petek po eni uri predavanj iz Biokemije.

Ocena seminarjev predstavlja 30% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev- datumi in seznam recenzentov še niso dokončni! ==
Vpišite svoj izbrani naslov!!!
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent2'''
|-
| Ula Štok||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 Tipping the mind]||17.10.11||19.10.11||21.10.11||Maja Remškar||Mirjam Kmetič
|-
| Maša Mirković||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 The twisted way of things]||17.10.11||19.10.11||21.10.11||Eva Knapič||Marko Radojković
|-
| Sara Draščič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 On the spur of a whim ]||17.10.11||19.10.11||21.10.11||Matevž Merljak||Monika Škrjanc
|-
| Katra Koman||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 Protein of the 20th century]||18.10.11||23.10.11||26.10.11||Ines Kerin||Veronika Jarc
|-
| Ana Dolinar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Ana_Dolinar:_Univerzalna_kri_.E2.80.93_prihodnost_transfuzijske_medicine.3F The juice of life]||21.10.11||25.10.11||28.10.11||Tjaša Goričan||Andreja Bratovš
|-
| Urška Rauter||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Ur.C5.A1ka_Rauter:_A_Green_Glow:_zgradba_in_funkcija_encima_luciferaze A green glow]||21.10.11||25.10.11||28.10.11||Maša Mohar||Sandi Botonjić
|-
| Taja Karner||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Taja_Karner:_Glavoboli_in_migrene Throb]||21.10.11||26.10.11||02.11.11||Karmen Hrovat||Tamara Marić
|-
| Rok Štemberger||Forbidden fruit||21.10.11||28.10.11||04.11.11||Špela Pohleven||Maja Grdadolnik
|-
| Maša Mohar||The tenuous nature of sex||21.10.11||28.10.11||04.11.11||Andreja Bratovš||Ines Kerin
|-
| Veronika Jarc||Our hollow architecture||21.10.11||28.10.11||04.11.11||Sabina Mavretič||Matevž Ambrožič
|-
| Mirjam Kmetič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Mirjam_Kmeti.C4.8D:_Mint_condition_.28limonen-3-hidroksilaza_in_limonen-6-hidroksilaza.29 Mint condition]||26.10.11||02.11.11||09.11.11||Sandi Botonjić||Tina Gregorič
|-
| Janez Meden||The Japanese Horseshoe Crab and Deafness||28.10.11||01.12.11||20.1.12||Veronika Jarc||Ana Dolinar
|-
| Tjaša Flis||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Sandi_Botonji.C4.87:_Kokain_esteraza Life's tremors]||28.10.11||04.11.11||11.11.11||Ana Dolinar||Špela Pohleven
|-
| Sandi Botonjić||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Sandi_Botonji.C4.87:_Kokain_esteraza Nature's junkie]||28.10.11||04.11.11||11.11.11||Maša Mirković||Alenka Mikuž
|-
| Kaja Javoršek||A grey matter||02.11.11||09.11.11||16.11.11||Dominik Kert||Tjaša Flis
|-
| Rok Vene||A mind astray||04.11.11||11.11.11||18.11.11||Tamara Marić||Maja Remškar
|-
| Ines Šterbal||One beer please||04.11.11||11.11.11||18.11.11||Ula Štok||Rok Vene
|-
| Matja Zalar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matja_Zalar:_Vloga_SRK_in_SCR_proteinov_pri_prepre.C4.8Devanju_incestnega_razmno.C5.BEevanja_c Do it yourself]||04.11.11||11.11.11||18.11.11||Monika Škrjanc||Matevž Merljak
|-
| Matevž Ambrožič||Of fidgets and food||09.11.11||16.11.11||23.11.11||Kaja Javoršek||Petra Malavašič
|-
| Matevž Merljak||Protein wars||11.11.11||18.11.11||25.11.11||Teja Banič||Urška Navodnik
|-
| Mitja Crček||When your day draws to an end||11.11.11||18.11.11||25.11.11||Marko Radojković||Andrej Vrankar
|-
| Dominik Kert||Talking heads||11.11.11||18.11.11||25.11.11||Alja Zottel||Kaja Javoršek
|-
| Petra Malavašič||Going unnoticed||16.11.11||23.11.11||30.11.11||Maja Grdadolnik||Mitja Crček
|-
| Eva Knapič||Life\'s first breath||18.11.11||25.11.11||02.12.11||Mirjam Kmetič||Andrej Vrankar
|-
| Marko Radojković||Paint my thoughts||18.11.11||25.11.11||02.12.11||Sara Draščič||Urška Rode
|-
| Tjaša Goričan||Nerve regrowth: nipped by a no-go||18.11.11||25.11.11||02.12.11||Ana Remžgar||Ines Šterbal
|-
| Tina Gregorič||Gut feelings||23.11.11||30.11.11||07.12.11||Janez Meden||Urška Rauter
|-
| Tamara Marić||The dark side of RNA||25.11.11||02.12.11||09.12.11||Dominik Kert||Rok Štemberger
|-
| Ana Remžgar||I'll have you for supper||25.11.11||02.12.11||09.12.11||Jana Verbančič||Eva Knapič
|-
| Maja Remškar||Questioning Colour||25.11.11||02.12.11||09.12.11||Katra Koman||Karmen Belšak
|-
| Andreja Bratovš||The power behind pain||30.11.11||07.12.11||14.12.11||Matevž Ambrožič||Teja Banič
|-
| Urška Navodnik||Darwin\'s dessert||02.12.11||09.12.11||16.12.11||Taja Karner||Karmen Hrovat
|-
| Jernej Mustar||Silent pain||02.12.11||09.12.11||16.12.11||Petra Malavašič||Jana Verbančič
|-
| Ines Kerin||A queen\'s dinner||02.12.11||09.12.11||16.12.11||Tjaša Flis||Iza Ogris
|-
| Alja Zottel||Sleepless nights||07.12.11||14.12.11||21.12.11||Ines Šterbal||Katra Koman
|-
| Alenka Mikuž||Molecular chastity||09.12.11||16.12.11||23.12.11||Urška Rode||Janez Meden
|-
| Maja Grdadolnik||Ear of Stone||09.12.11||16.12.11||23.12.11||Tina Gregorič||Ana Potočnik
|-
| Jana Verbančič||A balanced mind||09.12.11||16.12.11||23.12.11||Alenka Mikuž||Ana Remžgar
|-
| Karmen Hrovat||The thread of life||14.12.11||21.12.11||04.01.12||Iza Ogris||Taja Karner
|-
| Andrej Vrankar||The things we forget||16.12.11||23.12.11||06.01.12||Jernej Mustar||Maša Mohar
|-
| Teja Banič||Cool news||16.12.11||23.12.11||06.01.12||Karmen Belšak||Jernej Mustar
|-
| Špela Pohleven||The making of crooked||16.12.11||23.12.11||06.01.12||Mitja Crček||Maša Mirković
|-
| Sabina Mavretič||A short story||21.12.11||04.01.12||11.01.12||Rok Vene||Sabina Mavretič
|-
| Karmen Belšak||Another dark horse||23.12.11||06.01.12||13.01.12||Urška Rauter||Sara Draščič
|-
| Iza Ogris||Love,love, love...||23.12.11||06.01.12||13.01.12||Ana Potočnik||Matja Zalar
|-
| Monika Škrjanc||The greenest of us all||23.12.11||06.01.12||13.01.12||Rok Štemberger||Tjaša Goričan
|-
| Ana Potočnik||Skin-deep||04.01.12||11.01.12||18.01.12||Matja Zalar||Ula Štok
|-
| Urška Rode||Smart sweat||06.01.12||13.01.12||20.01.12||Urška Navodnik||Alja Zottel
|-
| Ime in priimek||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|}

== Gradivo za seminarje ==
NOVO Gradivo za predavanja in seminarje najdete na http://bio.ijs.si/~zajec/bio2/
username: bio2
password: samozame

==Naloga==
'''Vaša naloga za seminar je: '''
Samostojno pripraviti seminar o enem od proteinov opisanih v [http://web.expasy.org/spotlight/back_issues/2011/ ProteinSpotlight] Poiskati morate vsaj še tri znanstvene članke, ki se nanašajo na opisano temo in jih uporabiti kot podlago za seminarsko nalogo!

V okviru seminarske naloge morate opraviti še naslednje naloge, katerih rešitve predložite na dodatni strani seminarske naloge, ki se ne šteje v kvoto obsega seminarja:

* sekvenca proteina in [http://www.uniprot.org/ UniProt] oznaka proteina
* slika strukture proteina (če je le-ta znana), ki jo naredite sami s programom Pymol. Če struktura še ni znana, vključite sliko proteina, ki je vašemu najbolj podoben po sekvenci in katerega struktura je znana
* poiskati morate, na katerem kromosomu se v človeškem genu nahaja ta protein in narisati shematsko sliko gena (eksonov in intronov) tega proteina. Če protein ni človeškega izvora, poiščite protein, ki je vašemu najbolj podoben in vse navedeno opišite za ta protein.

Za pripravo seminarja velja naslednje: 
* [[BIO2 Povzetki seminarjev 2011|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge na ~5-9 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 2700 do 3000 besed), vsebovati mora najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Seminar oddajte do datuma oddaje, ki je naveden v tabeli vsakemu od recenzentov in docentu (docentu ga pošljite po e-pošti).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20-30 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo oceno predstavitve in postavijo najmanj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [https://docs.google.com/spreadsheet/viewform?formkey=dG1Pa3p2NXE2Vm1zX3FpVTZCT2dHVnc6MA recenzentsko poročilo] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://docs.google.com/spreadsheet/viewform?formkey=dFNXUDBCRVBaVExvOFVxakpJUHRnOEE6MA mnenje] najkasneje v šestih dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO2 Povzetki seminarjev 2011

2011-11-04T13:31:55Z

MatjaZalar:

== Sara Draščič: On the spur of a whim ==

Serotonin ali 5-hidroksitriptamin (5-HT) spada v skupino heterogenih biokemičnih snovi, ki prenašajo informacije po živčnem sistemu in ki jim rečemo nevrotransmiterji. Ima pomembno vlogo pri veliko najrazličnejših reakcijah v telesu. Njegovo nepravilno delovanje vpliva na počutje, apetit, slabost, spanje, telesno temperaturo, staranje, bolečino, anksioznost, agresijo, spomin, migrene in na številne druge procese v organizmu. Večina serotonina se sintetizira v prebavnem traktu, preostali del pa v centralnem živčnem sistemu in trombocitih. Kljub temu, da se sintetizira le v določenih delih telesa, je prisoten povsod. Dokaz za njegovo prisotnost pa so serotoninski receptorji. Serotonin ima veliko receptorjev, ki so jih organizirali v sedem skupin glede na njihove fiziološke in strukturne razlike. Ravno zaradi tako velikega števila raznoraznih receptorjev, je serotonin pomemben pri tolikih različnih procesih, saj je njegovo delovanje, v veliki meri, odvisno od tega, na kateri receptor se bo vezal. Veliki pomen pri delovanju serotonina ima tudi njegov transporter. To je protein, katerega struktura še ni znana, vendar vemo kje in na katerem kromosomu se nahaja. Transporter je tudi glavna tarča raznih antidepresivov in drog kot so ecstasy, kokain in LSD.

== Ula Štok: Neuregulin 1 ==

Neuregulin-1 je član proteinov iz družine neuregulinov in je kodiran s strani gena NRG1. Obstaja veliko tipov Neuregulina-1, ki se razlikujejo po funkcionalnosti ter mestu v telesu na katerem delujejo. Najpogosteje delujejo v živčnem sistemu, kjer lahko z nepravilnim delovanjem med drugimi povzročajo tudi zelo razširjeno bolezen - shizofrenijo. Delujejo pa tudi na ostalih tkivih in organih (na primer: srce, pljuča, oprsje in želodec). Generalno obstajata dve poti signaliziranja Neuregulina-1, in sicer: Običajna ter neobičajna pot. Pri običajni poti je ErbB receptor aktiviran direktno, v enem koraku z vezavo Neuregulina-1. To najpogosteje povzroči dimerizacijo ali heterodimerizacijo ErbB receptorja. Dimerizacija ali heterodimerizacija sicer nista nujno potrebni, a vendar do njiju pride na skoraj vseh receptorjih ErbB. Ta združitev povzroči avto- in trans-fosforilacijo intracelularnih domen tega receptorja, kar aktivira vse nadaljnje poti signaliziranja. V končni fazi pa NRG1/ErbB signaliziranje vpliva direktno na transkripcijo. Pri neobičajni poti je postopek podoben, a vendar poteka začetna stopnja malo drugače. Na začetku namreč sodeluje JMa oblika receptorja ErbB4, ki se pod vplivom TACE cepi. Del receptorja (ErbB4-CTF) se odcepi v notranjost celice. Ta peptid je velik približno 80 kD in ima specifično izoblikovano vezavno mesto za Neuregulin-1. Nadaljnji procesi pa potekajo zelo podobno kot pri običajni signalni poti. Neuregulin-1 lahko povzroča shizofrenijo na različne načine, saj sodeluje pri zelo pomembnih procesih, kot so: tvorba sinaps, mielinizacija aksonov, razvoj oligodendrocit itd. Shizofrenija je zelo razširjena bolezen in nihče še ni odkril direktnega postopka k popolni odpravi te bolezni. A vendar, v letu 2009 se je zgodila neke vrste prelomnica v študiju shizofrenije. Odkrili so namreč, da posamezniki, ki so imeli gen za shizofrenijo niso zboleli. Še več! Napaka se jim je odrazila kot zvišanje kreativnih sposobnosti na znanstvenem ali umetniškem področju, odvisno od posameznika. Ob tem se je pojavilo mnogo vprašanj, saj bi na ta način mogoče lahko poiskali pot, da bi shizofrenija postala popolnoma ozdravljiva. A vendar, je to področje še raziskano, saj znanstveniki ne vedo po kakšnih poteh pride do tega, da te mutacije na NRG1 genu ne izrazijo v bolezenskem stanju.

== Maša Mirković: Proteinski produkti genov za disleksijo in z disleksijo povezane motnje ==

Disleksija je motnja, ki se kaže v nesposobnosti branja oziroma razumevanja prebranega, ter napakah in težavah pri izgovarjanju besed. Disleksiki,kot imenujemo posameznike, ki trpijo za disleksijo, imajo kljub normalnim intelektualnim sposobnostim, znanjem in izobrazbo, moteni veščini pisanja in branja s tendenco, da pomešajo med seboj črke ali besede med branjem ali pisanjem. V zadnjih letih, so uspeli ugotoviti mesta na kromosomih, povezana z dovzetnostjo za disleksijo. DYX1C1,KIAA0319,DCDC2 in ROBO1, so bili označeni kot kandidati, z dovzetnostjo za disleksijo. Najbolj obetaven je protein KIAA0319. Je transmembranski protein iz desetih transmembranskih vijačnic, najden v plazemski membrani nevronov. Njegov C-terminalni konec gleda v ekstracelularni matriks, manjši N-terminalni konec pa prehaja v citoplazmo nevrona. C-terminalni konec je visoko glikoziliran in nosi 5 PKD(polycystyc kidney desease) domene in eno MANEC(motif at the N terminus with eight cysteines) domeno. KIAA0319 igra vlogo pri rasti možganov in njihovi migraciji med razvojem možganov-iz tega je razvidno, da je disleksija problem v razvoju nevronov že v zgodnjih letih. Posamezniki z disleksijo nosijo izoobliko tega proteina, ki povzroči nižjo izraženost le tega. Spremembe so v 5'-regiji, ki kodira izoobliko proteina. Najopaznejše povezave z disleksijo se kažejo v 2,3 kb regiji, ki zavzema promotor, prvi nepreveden ekson in del prvega introna – odprti kromatin. Te ugotovitve vodijo, da je 5'-regija KIAA0319 gena tista lokacija alelov, ki največ prispeva k motnji branja.

== Katra Koman: INZULIN ==

Inzulin je peptidni hormon, ki sodeluje v uravnavanju ravni glukoze v krvi. Sintetizira in skladišči se v β-celicah Langerhansovih otočkov trebušne slinavke. Sinteza poteka od prekurzorske molekule preproinzulina preko proinzulina do dokončne zrele molekule inzulina, ki se shrani v skladiščnih veziklih. Ob povišanju ravni glukoze v krvi, na primer po obroku, glukoza, ki je tudi glavni stimulator sekrecije inzulina, iz krvi preide v β-celice skozi GLUT2 transporter. Tam se fosforilira v glukozo-6-fosfat, saj tako fosforilirana ne more več iz celice, lahko pa vstopi v proces glikolize, ki mu sledita še Krebsov cikel in oksidativna fosforilacija, ki povzroči pretvorbo ADP v ATP molekule. ATP molekula stimulira zaprtje kalijevih kanalčkov, kar privede do depolarizacije celične membrane, to pa sproži na odprtje kalcijevih kanalčkov in vdor Ca2+ ionov. Povišana koncentracija kalcijevih Ca2+ ionov v celici stimulira prenos in zlitje skladiščnih veziklov z inzulinom z membrano. Inzulin se tako sprosti v krvni obtok in potuje do tarčnih celic, ki imajo na površini izražene inzulinske receptorje. Ko se veže nanj, prenese signal o povišanju ravni glukoze v krvi v celico. To povzroči kaskado reakcij znotraj celice, ki pa na koncu privedejo do translokacije veziklov z GLUT4 transporterjev na površino celice. Število teh transporterjev za glukozo se na površini celične membrane poveča in glukoza lahko prehaja v celico, posledično pa pade raven glukoze v krvi. Razgradnja inzulina poteka v jetrih in ledvicah. Okvare na katerikoli stopnji poti inzulina se odražajo v diabetesu ali drugih boleznih.

== Rok Štemberger: Protein GABAA (gama aminomaslena kislina A) - zgradba, vloga in zanimivosti ==

V svoji seminarski nalogi sem raziskoval vlogo, pomen in zanimivosti proteina GABAA (gama-aminomaslena kislina A). To je receptor, ki se nahaja predvsem v centralnem živčnem sistemu in je zadolžen zato, da opravlja funkcijo inhibitorja. Lociran je na površini nevrotičnih sinaps in prekinja elektrokemični signal, tako da omogoči prehod kloridnih ionov znotraj celice. To se zgodi takrat ko se ustrezen ligand Gama veže na aktivno mesto tega receptorja. Konformacija podenot se spremeni in to omogoči aktivacijo receptorja. Znanstveniki so ugotovili, da obstaja več vrst GABAA receptorjev, kar pa je odvisno od sestave podenot. Najbolj pogoste podenote so alfa beta in gama v razmerju 2:2:1. V primeru da do prekinitve ne pride se lahko pojavijo epileptični napadi, psihiatrične motnje itd. Stres lahko v dobi odraščanja močno vpliva na GABAA receptorje in jih tudi permanentno strukturno spremeni, kar pa lahko kasneje v našem življenju vpliva predvsem na naš spanec in njegovo kvaliteto. Absint je bila v preteklosti prepovedana pijača, saj je povzročala razna obolenja zaradi substance imenovane tujon. Le ta se je vezala na GABAA receptorje in tako onemogočila njegovo delovanje, zato ker je preprečevala prehod kloridnih ionov v membrano. Sedaj potekajo raziskave teh receptorjev, saj je ključnega pomena čim boljša ozdravitev bolezni, ki nastanejo zaradi nepravilnega delovanja GABAA receptorja.

== Veronika Jarc: Perforin ==

Perforin je protein, ki nastane iz citotoksičnih limfocitov T. S pomočjo grancimov napade tarčno celico in jo uniči. Rečemo lahko, da je pomemben člen pri imunskem odzivu in sodeluje s NK celicami. Sestavljen je iz 555 aminokislin, njegova molekulska masa pa je 62-67 kD. Sestavljen je iz dveh pomembnih domen, domene MACPF in domene C2. Za domeno C2 je značilno, da ima afiniteto do Ca2+ ionov. Saj se na lipidni dvosloj veže le ob prisotnosti kalcija. Drugače obstajata dva različna tipa C2 domene, ki sta bila izolirana iz različnih organizmov. Lahko rečemo, da sta oba tipa zelo podobna v tem, da sta pri tipu 1 N-konec in C-konec obrnjena na vrh domene, kar je nasprotno kot pri tipu 2. Poznamo tri MACPF domene: Plu-MACPF, C8a MACPF in lipokalin C8g. Vse te domene primerjamo z skupino proteinov citolizinov in ugotovimo nekaj podobnosti in nekaj razlik. Na splošno, pa lahko rečemo, da je evolucija poskrbela tako, da so sta si domena MACPF in citolizini raszlični le v nekaj aminokislinah. Poznamo tri mehanizme kako perforin preide v tarčno celico in pri tem pomaga gramcimom B uničit to celico. Prvi mehanizem je prehajanje preko perforinske pore in sicer s pomočjo veziklov preide v celico. Naslednji mehanizem je endosomolitični model, pri katerem je pomemben kompleks s pomočjo katerega prehaja v celico. Kot zadnji mehanizem pa je model prehodne perforinske pore, ki pove, da perforin tvori kanalčke s pomočjo katerih grancimi B preidejo direktno v celico. Grancimi B so serinske proteaze, ki se sintetizirajo v citotoksičnih limfocitih T in NK celicah.

== Taja Karner: Glavoboli in migrene ==

Zaradi stresnega in hitrega tempa življenja, vse več ljudi trpi za občasnimi glavoboli, ki so najpogosteje posledica utrujenosti. Prav tako je vedno več ljudi, ki trpijo za močnejšimi oblikami glavobolov imenovanih migrene. V hujših oblikah migrene lahko glavobol traja do dva dni, močno migreno lahko spremljajo še drugi simptomi kot so slabost, bruhanje, občutljivost na svetlobo in močan zvok, depresija ter nespečnost. Mutacija, ki je največji krivec za nastanek bolezni se pojavlja na kromosomu 10 na genu KCNK18. Ta zapisuje protein TRESK, ki se nahaja v hrbtenjači in deluje kot kalijev kanalček. Mutacija povzroči, da ne pride do izmenjavanja ionov, kar povzroči hude glavobole. V raziskavah so odkrili zanimivo povezavo z anestetikom. Ta namreč ne glede na mutacijo ponovno aktivira kanal. To bi lahko učinkovito pozdravilo migrene, če bi ga le uspeli spraviti v primerno obliko. Ugotovili so tudi, da zdravila, ki vsebujejo citosporin in takrolimus v večini primerov povzročajo migrene v zdravstvu pa jih še vseeno pogosto uporabljajo. Odkritje te mutacije predstavlja revolucijo v zdravstvu in verjamem, da bo kmalu vodilo do odkritja učinkovitega zdravila proti migrenam.

== Ana Dolinar: Univerzalna kri – prihodnost transfuzijske medicine? ==

α-galaktozidaza (AGAL_HUMAN) je glikozil-hidrolazni encim. Spada v GH27-D (klan D, 27. družina) in ima aktivno mesto v obliki (β/α)8 sodčka. Encim zapisuje gen GLA, ki se nahaja na kromosomu X.

Ideja o univerzalni krvi, ki bi bila primerna za transfuzijo, ne glede na krvno skupino pacienta, je med znanstveniki prisotna že približno trideset let.
Razvili so tri metode za pretvorbo različnih antigenov v antigen 0 (po sistemu AB0), ki je primeren za transfuzijo v vse krvne skupine.
:#Encimska razgradnja antigenov A in B do antigena 0. Za antigene A so uporabili α-N-acetilgalaktozaminidazo, vendar so antigeni preveč kompleksni in metoda ni bila uspešna. Pri antigenih B so dosegli popolno pretvorbo v antigen 0 z uporabo α-galaktozidaze iz bakterije ''Streptomyces griseoplanus''.
:#Prekrivanje površine eritrocitov z maleimidofenil-polietilen-glikolom (Mal-Phe-PEG). Prekrije vse antigene, ne samo A ali B, vendar metoda ni uspešna, ker polietilen-glikol povzroča imunski odziv.
:#Pridobivanje univerzalnih rdečih krvnih celic iz pluripotentnih matičnih celic. Uspeli so pridobiti zrele eritrocite, ki so popolnoma funkcionalni.
Uporaba univerzalne krvi bi zmanjšala ali celo izničila imunski odziv ob transfuziji, prav tako ne bi bilo možnosti za transfuzijo napačne krvne skupne zaradi človeške napake. Metode trenutno niso dovolj izpopolnjene, da bi bilo možno pričakovati njeno uporabo v bližnji prihodnosti.

== Maša Mohar: Moški ali ženska to je sedaj vprašanje?(SRY - faktor za določitev spola) ==

SRY gen kodira Sry protein ki je član družine Sox (Sry related HMG box) transkripcijskih faktorjev. Poznamo jih okoli 20 pri človeku in miškah ter še mnogo drugih. Sox proteini imajo zelo različne vloge v embriogenezi in pri razvoju mnogih drugih organov. Tipično delujejo tako kot nekakšna stikala v diferenciaciji celic- sprožijo razvoj določenih celic. Sry je prav tako kot ostali člani te družine karakteriziran po HMG( high mobility group). HMG je drugače skupina specifičnih transkripcijskih faktorjev, ki imajo ~ 80 AK dolge strukturalno podobne domene za vezavo na DNA. Te domene oz. domena če je samo ena se veže na zaporedje (A/T)ACAA(T/A) v majhni žleb DNA. S tem ustvari zvitje DNA za približno 60- 85 stopinj. S tem ko se DNA zvije se razkrijejo mesta za izražanje drugih genov, recimo Sox9, ki kodira Sox9 protein ki pomaga pri diferenciaciji Sertoli celic in tako pri oblikovanju testisov, s tem pa determinira moški spol. Ugotovili smo tudi da obstaja veliko genskih bolezni povezanih s Sry genom in da lahko obstaja tudi ženska z XY spolnima kromosomoma, ker se pri njej zaradi mutacij Sry protein ne izrazi, prav tako pa obstajajo tudi moški z XX spolnima koromosomoma, kjer se enem od X kromosomov lahko izrazi SRY gen ob nepravilnostih pri očetovem delu zapisa. V bistvu sem prišla do zaključka da je zelo tanka meja med moškim in ženskim oblikovanjem spola, ena majhna mutacija oz. ena majhna razlika lahko privede do nastanka ženske ali moškega.

== Urška Rauter: A Green Glow: zgradba in funkcija encima luciferaze ==
Luciferaza je encim odvisen od ATP in magnezijevih ionov. Proces bioluminiscence se začne z vezavo na substrat luciferin, tvori se adenilatni intermediat in ob prisotnosti molekularnega kisika izhaja svetloba. Luciferaza je zgrajena iz dveh ločenih domen, večja se nahaja na N-koncu in manjša na C-koncu molekule, večja domena pa ima tudi svoje poddomene. Domeni sta med seboj ločeni z razpoko, kjer naj bi se po domnevanjih nahajalo tudi aktivno mesto encima. Luciferaza predstavlja tudi nov način mehanizma tvorbe adenilatnega intermediata med encimi in ponuja razlago za marsikatero metabolično pot.
Velika dilema, ki me med znanstveniki ostaja pa je razlika v barvi svetlobe, ki jo proces oksidacije luciferina emitira. Najverjetneje je za to odločilna keto tavtomerna oblika oksiluciferina in tudi resnonančna stabilizacija njegovega fenolatnega aniona, čeprav so znanstveniki odkrili tudi veliko drugih možnih vzrokov za različne barve (različne aminokisline, polarnost okolja, pH, ...).
Luciferaza se veliko uporablja v medicini, kjer služi kot marker molekul v telesu in tako pripomore k boljšem razumevanju različnih bolezni in infekcij, kot tudi sami strukturi celic in njenih organelov.

== Mirjam Kmetič: Mint condition (limonen-3-hidroksilaza in limonen-6-hidroksilaza) ==

Klasasta meta vsebuje encim limonen-6-hidroksilazo, ki sodeluje pri pridobivanju karvona. Poprova meta pa vsebuje limonen-3-hidroksilazo, ki je udeležena pri proizvodnji mentola. Obe hidroksilazi pripadata družini citokromov P450, njeni predstavniki pomembno sodelujejo pri proizvajanju različnih oksidiranih monoterpenov, ki so vir arom eteričnih olj. Karvon in mentol sta končna produkta hidroksilacije limonena. Ta encima sta si zelo podobna in njuni vezavni mesti za substrat sta zelo omejeni. Velja pravilo, da za spremembo aktivnosti v družini citokromov P450 potrebujemo določeno število mutacij, vendar je za modifikacijo vezavne aktivnosti limonenovih hidroksilaz potrebna samo ena. Ta fenilalanin v izolevcin mutacija povzroči, da se limonen-6-hidroksilaza spremeni v limonen-3-hidroksilazo! Mutiran encim je tako sposoben sinteze mentola tako kot encim v poprovi meti! Taka mutacija kaže, da sta prav ti dve aminokislini ne le nujni, temveč tudi prav zagotovo vpleteni pri orientaciji limonena v aktivnem mestu tako, da se ta hidroksilizira na ali C3 ali C6 poziciji. Posamične mutacije, ki lahko drastično spremenijo funkcijo proteina, so znanstveno zanimive. Nakazujejo ne le na zelo specifične manjše regije v sekvenici proteina, temveč so tudi ključne za razumevanje področij, kot so vezava in orientacija substrata, funkcija encima, metabolična pot in struktura vezavnega mesta.

== Sandi Botonjić: Kokain esteraza ==

Znanstveniki so v rizosferi kokinih plantaž (Erythroxylum coca) našli sev MB1, gram pozitivne bakterije Rhodococcus sp.. Tej bakteriji kokain predstavlja glavni vir ogljika in dušika in zato so znanstveniki izolirali osrednji encim njenega metabolizma tj. kokain esterazo (v nadaljevanju cocE). Encim je sestavljen iz treh domen: DOM1, ki vsebuje nabor kanoničnih α-vijačnic in β-ploskev; DOM2 - domena le z α-vijačnicami; in DOM3 je roladi podobna struktura z β-ploskvami. CocE je serinska esteraza, katere aktivno mesto se nahaja na stičišču vseh treh domen. Ta hidrolizira kokain na ekgonil metil ester in benzojsko kislino, ki nimata psihoaktivnih učinkov. CocE je pravi Ferrari v primerjavi z drugimi esterazami, saj lahko razgradi enako količino kokaina 1000 krat hitreje. Tako lahko postane neprecenljiva pri nujnih intervencijah v primeru prevelikega odmerka, saj bi intravenozni vbrizg cocE močno zmanjšal razpolovni čas kokaina. CocE je predmet številnih raziskav, v katerih znanstveniki proučujejo njeno termostabilnost in njenih mutiranih oblik, saj njen razpolovni čas pri fiziološki temperaturi traja le nekaj minut. Znanstveniki pa na podlagi ugotovitev iz raziskav cocE razvijajo tudi učinkovita protitelesa z vsaj podobnimi katalitičnimi parametri, ki bi brez imunskega odziva odlično delovala v bioloških sistemih.

==Tjaša Flis: Parkinsonizem in Parkin protein==

Parkinsonova bolezen je vse pogostejša bolezen pri starostnikih, njeni simptomi pa so tresavica, mišična otrdelost in upočasnjena motorika. Vzrok se skriva v propadu dopamnergičnih nevronskih celic. Bolezen je lahko avtosomno dominantno dedovana, kar pomeni, da pacienti podedujejo eno normalno in eno mutirano kopijo gena. Slednja prevladuje in se deduje naprej. Pri Parkinsonovi bolezni se mutacija zgodi v Park2 genu, ki kodira Parkin protein ali E3 ubikvitin ligazo. Parkin na poškodovane ali na preveč izražene proteine pripne ubikvitin (označevalni protein), ki jih nato usmeri v proteasom, to je velik razgradni kompleks v celicah.
Če mutacija poškoduje Parkin, je pot razgradnje onemogočena, to pa pomeni, da se v celici akumulirajo odvečni proteini. Tvorijo se Lewy-eva telesca polna teh proteinov, ki nadomestijo celične organele v nevronskih celicah, kar vodi do prenehanja njihovega delovanja. Ker pa ima Parkin več kot samo en substrat ki ga ubikvitinira, je točen mehanizem bolezni še dandanes uganka.
Eden izmed najbolj poznanih substratov je transmembranski protein Pael-R. Zvitje tega proteina poteka ob prisotnosti šaperonov. Prevelika koncentracija tega receptorja lahko izzove stres v endoplazmatskem retikulumu situiranem v nevronskih celicah. V primeru da je Parkin neaktiven, Pael-R povzroči celično smrt. Vendar to je le ena izmed možnih rešitev, substratov je namreč vsaj še dvajset, raziskave pa se nadaljujejo.

== Matja Zalar: Vloga SRK in SCR proteinov pri preprečevanju incestnega razmnoževanja cvetočih rastlin ==

Rastline so za zaščito pred samooplojevanjem razvile več vrst mehanizmov prepoznavanja lastnega peloda na molekularni ravni. Pri cvetočih rastlinah je najpogostejši mehanizem tipa SSI ali sporofitične lastne inkompatibilnosti. Pri družini ''Brassicaceae'' je za aktivacijo SSI ključna interakcija med transmembranskim proteinom SRK, ki predstavlja žensko determinanto odziva, in njenim ligandom - proteinom SCR, drugače imenovanim tudi moška determinanta odziva na lastno inkompatibilnost. Specifičnost vezave je zagotovljena s polimorfizmom alel obeh determinant. V posameznih vrstah je možno najti tudi do 100 različnih S-haplotipov genov za determinanti.
Vezava liganda na receptor bo uspešna le, če oba izhajata iz istega S-haplotipa. Vezava SCR na zunajcelično, N-glikolizirano domeno SRK povzroči nastanek kompleksa treh proteinov, ki s svojo aktivnostjo sproži kaksado reakcij, kar v končni fazi pripelje do preprečitve samooploditve.
Na neugodne življenske pogoje, ki so onemogočali medsebojno opraševanje, so se nekatere rastline prilagodile s favorizacijo samooplojevanja. Pri njih so mutacije S-lokusa, ki nosi zapis za SRK in SCR, povzročile nepravilno delovanje SI ali njegovo popolno odpoved. To pa seveda vodi v neprepoznavanje lastnega peloda in rastlina se samooprašuje. Najbolj znan primer take rastline je ''Arabidopsis thaliana'', ki se zaradi svojih specifičnih lastnosti uporablja kot modelni organizem v številnih študijah lastne inkompatibilnosti.

BIO2 Povzetki seminarjev 2011

2011-10-17T19:54:12Z

MatjaZalar:

Serotonin ali 5-hidroksitriptamin (5-HT) spada v skupino heterogenih biokemičnih snovi, ki prenašajo informacije po živčnem sistemu in ki jim rečemo nevrotransmiterji. Ima pomembno vlogo pri veliko najrazličnejših reakcij v telesu. Njegovo nepravilno delovanje vpliva na počutje, apetit, slabost, spanje, telesno temperaturo, staranje, bolečino, anksioznost, agresijo, spomin, migrene in na številne druge procese v organizmu. Večina serotonina se sintetizira v prebavnem traktu, preostali del pa v centralnem živčnem sistemu in trombocitih. Kljub temu, da se sintetizira le v določenih delih telesa, je prisoten povsod. Dokaz za njegovo prisotnost pa so serotoninski receptorji. Serotonin ima veliko receptorjev, ki so jih organizirali v sedem skupin glede na njihove fiziološke in strukturne razlike. Ravno zaradi tako velikega števila raznoraznih receptorjev, je serotonin pomemben pri tolikih različnih procesih, saj je njegovo delovanje, v veliki meri, odvisno od tega na kateri receptor se bo vezal. Veliki pomen pri delovanju serotonina ima tudi njegov transporter. To je protein, katerega struktura še ni znana, vendar vemo kje in na katerem kromosomu se nahaja. Transporter je tudi glavna tarča raznih antidepresivov in drog kot so ecstasy, kokain in LSD.

Ula Štok: Neuregulin 1

Neuregulin-1 je član proteinov iz družine neuregulinov in je kodiran s strani gena NRG1. Obstaja veliko tipov Neuregulina-1, ki se razlikujejo po funkcionalnosti ter mestu v telesu na katerem delujejo. Najpogosteje delujejo v živčnem sistemu, kjer lahko z nepravilnim delovanjem med drugimi povzročajo tudi zelo razširjeno bolezen - shizofrenijo. Delujejo pa tudi na ostalih tkivih in organih (na primer: srce, pljuča, oprsje in želodec). Generalno obstajata dve poti signaliziranja Neuregulina-1, in sicer: Običajna ter neobičajna pot. Pri običajni poti je ErbB receptor aktiviran direktno, v enem koraku z vezavo Neuregulina-1. To najpogosteje povzroči dimerizacijo ali heterodimerizacijo ErbB receptorja. Dimerizacija ali heterodimerizacija sicer nista nujno potrebni, a vendar do njiju pride na skoraj vseh receptorjih ErbB. Ta združitev povzroči avto- in trans-fosforilacijo intracelularnih domen tega receptorja, kar aktivira vse nadaljnje poti signaliziranja. V končni fazi pa NRG1/ErbB signaliziranje vpliva direktno na transkripcijo. Pri neobičajni poti je postopek podoben, a vendar poteka začetna stopnja malo drugače. Na začetku namreč sodeluje JMa oblika receptorja ErbB4, ki se pod vplivom TACE cepi. Del receptorja (ErbB4-CTF) se odcepi v notranjost celice. Ta peptid je velik približno 80 kD in ima specifično izoblikovano vezavno mesto za Neuregulin-1. Nadaljnji procesi pa potekajo zelo podobno kot pri običajni signalni poti. Neuregulin-1 lahko povzroča shizofrenijo na različne načine, saj sodeluje pri zelo pomembnih procesih, kot so: tvorba sinaps, mielinizacija aksonov, razvoj oligodendrocit itd. Shizofrenija je zelo razširjena bolezen in nihče še ni odkril direktnega postopka k popolni odpravi te bolezni. A vendar, v letu 2009 se je zgodila neke vrste prelomnica v študiju shizofrenije. Odkrili so namreč, da posamezniki, ki so imeli gen za shizofrenijo niso zboleli. Še več! Napaka se jim je odrazila kot zvišanje kreativnih sposobnosti na znanstvenem ali umetniškem področju, odvisno od posameznika. Ob tem se je pojavilo mnogo vprašanj, saj bi na ta način mogoče lahko poiskali pot, da bi shizofrenija postala popolnoma ozdravljiva. A vendar, je to področje še raziskano, saj znanstveniki ne vedo po kakšnih poteh pride do tega, da te mutacije na NRG1 genu ne izrazijo v bolezenskem stanju.

BIO2 Seminar 2011

2011-10-09T20:54:38Z

MatjaZalar: /* Seznam seminarjev - datumi še niso dokončni, listka na katerem imam napisano kdaj kdo ne more nimam doma in bom to popravil v ponedeljek */

= Biokemijski seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsako sredo in petek po eni uri predavanj iz Biokemije.

Ocena seminarjev predstavlja 30% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev - datumi še niso dokončni, listka na katerem imam napisano kdaj kdo ne more nimam doma in bom to popravil v ponedeljek==
Vpišite svoj izbrani naslov!!!
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent2'''
|-
| Ula Štok||Tipping the mind||17.10.11||19.10.11||21.10.11||||
|-
| Maša Mirković||Naslov seminarja||17.10.11||19.10.11||21.10.11||||
|-
| Sara Draščič||On the spur of a whim||17.10.11||19.10.11||21.10.11||||
|-
| Katra Koman||Naslov seminarja||18.10.11||23.10.11||26.10.11||||
|-
| Iza Ogris||Naslov seminarja||21.10.11||25.10.11||28.10.11||||
|-
| Ana Remžgar||Naslov seminarja||21.10.11||25.10.11||28.10.11||||
|-
| Urška Rauter||Naslov seminarja||21.10.11||25.10.11||28.10.11||||
|-
| Taja Karner||Throb||21.10.11||26.10.11||02.11.11||||
|-
| Rok Štemberger||Forbidden fruit||21.10.11||28.10.11||04.11.11||||
|-
| Maša Mohar||The tenuous nature of sex||21.10.11||28.10.11||04.11.11||||
|-
| Veronika Jarc||Our hollow architecture||21.10.11||28.10.11||04.11.11||||
|-
| Mirjam Kmetič||Naslov seminarja||26.10.11||02.11.11||09.11.11||||
|-
| Janez Meden||Naslov seminarja||28.10.11||04.11.11||11.11.11||||
|-
| Tjaša Flis||Naslov seminarja||28.10.11||04.11.11||11.11.11||||
|-
| Sandi Botonjić||Naslov seminarja||28.10.11||04.11.11||11.11.11||||
|-
| Kaja Javoršek||Naslov seminarja||02.11.11||09.11.11||16.11.11||||
|-
| Rok Vene||Naslov seminarja||04.11.11||11.11.11||18.11.11||||
|-
| Ines Šterbal||Naslov seminarja||04.11.11||11.11.11||18.11.11||||
|-
| Andreja Bratovš||The power behind pain||04.11.11||11.11.11||18.11.11||||
|-
| Matevž Ambrožič||Naslov seminarja||09.11.11||16.11.11||23.11.11||||
|-
| Matevž Merljak||Naslov seminarja||11.11.11||18.11.11||25.11.11||||
|-
| Mitja Crček||Naslov seminarja||11.11.11||18.11.11||25.11.11||||
|-
| Dominik Kert||Naslov seminarja||11.11.11||18.11.11||25.11.11||||
|-
| Petra Malavašič||Going unnoticed||16.11.11||23.11.11||30.11.11||||
|-
| Eva Knapič||Life's first breath||18.11.11||25.11.11||02.12.11||||
|-
| Marko Radojković||Naslov seminarja||18.11.11||25.11.11||02.12.11||||
|-
| Tjaša Goričan||Nerve regrowth: nipped by a no-go||18.11.11||25.11.11||02.12.11||||
|-
| Tina Gregorič||Naslov seminarja||23.11.11||30.11.11||07.12.11||||
|-
| Tamara Marić||The dark side of RNA||25.11.11||02.12.11||09.12.11||||
|-
| Ana Dolinar||The juice of life||25.11.11||02.12.11||09.12.11||||
|-
| Maja Remškar||Naslov seminarja||25.11.11||02.12.11||09.12.11||||
|-
| Matja Zalar||Do it yourself||30.11.11||07.12.11||14.12.11||||
|-
| Urška Navodnik||Naslov seminarja||02.12.11||09.12.11||16.12.11||||
|-
| Jernej Mustar||Silent pain||02.12.11||09.12.11||16.12.11||||
|-
| Ines Kerin||A queen's dinner||02.12.11||09.12.11||16.12.11||||
|-
| Alja Zottel||Sleepless nights||07.12.11||14.12.11||21.12.11||||
|-
| Alenka Mikuž||Molecular chastity||09.12.11||16.12.11||23.12.11||||
|-
| Maja Grdadolnik||Ear of Stone||09.12.11||16.12.11||23.12.11||||
|-
| Jana Verbančič||Hidden power||09.12.11||16.12.11||23.12.11||||
|-
| Petra Gorečan||Naslov seminarja||14.12.11||21.12.11||04.01.12||||
|-
| Karmen Hrovat||Naslov seminarja||16.12.11||23.12.11||06.01.12||||
|-
| Andrej Vrankar||The things we forget||16.12.11||23.12.11||06.01.12||||
|-
| Teja Banič||Cool news||16.12.11||23.12.11||06.01.12||||
|-
| Špela Pohleven||Naslov seminarja||21.12.11||04.01.12||11.01.12||||
|-
| Sabina Mavretič ||A short story||23.12.11||06.01.12||13.01.12||||
|-
| Karmen Belšak ||Another dark horse||23.12.11||06.01.12||13.01.12||||
|-
| Ime in priimek ||Naslov seminarja||23.12.11||06.01.12||13.01.12||||
|-
| Ime in priimek ||Naslov seminarja||04.01.12||11.01.12||18.01.12||||
|-
| Ime in priimek ||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|-
| Ime in priimek ||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|-
| Ime in priimek ||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|}

== Gradivo za seminarje ==
Gradivo za predavanja in seminarje najdete na http://bio.ijs.si/~zajec/bio2/
username: bio2
password: samozame

==Naloga==
'''Vaša naloga za seminar je: '''
Samostojno pripraviti seminar o enem od proteinov opisanih v [http://web.expasy.org/spotlight/back_issues/2011/ ProteinSpotlight] Poiskati morate vsaj še tri znanstvene članke, ki se nanašajo na opisano temo in jih uporabiti kot podlago za seminarsko nalogo!
V seminarsko nalogo mora biti vključeno:
* sekvenca proteina in SwissProt oznaka proteina
* slika strukture proteina (če je le-ta znana), ki jo naredite sami s programom Pymol. Če struktura še ni znana, vključite sliko proteina, ki je vašemu najbolj podoben po sekvenci in katerega struktura je znana
* poiskati morate, na katerem kromosomu se v človeškem genu nahaja ta protein in narisati shematsko sliko gena (eksonov in intronov) tega proteina. Če protein ni človeškega izvora, poiščite protein, ki je vašemu najbolj podoben in vse navedeno opišite za ta protein.

Za pripravo seminarja velja naslednje: 
* [[BIO2 Povzetki seminarjev 2011|Povzetek seminarja]] opišete na wikiju v približno 200 besedah, besedilo naj vsebuje sliko strukture proteina, ki jo sami narišete s programom PyMol - najkasneje do dne ko morate oddati seminar recenzentom.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge na ~5-9 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 2700 do 3000 besed), vsebovati mora najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Natisnjen seminar oddajte dva tedna pred predstavitvijo vsakemu od recenzentov (docentu ga pošljite po e-pošti v formatu .doc ali .docx).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20-30 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo oceno predstavitve in postavijo najmanj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [[https://spreadsheets.google.com/viewform?hl=en&formkey=dE1aOFU1aE1iMlBrNEJzLTRGeTdWZXc6MQ#gid=0 recenzentsko poročilo]] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://spreadsheets.google.com/viewform?hl=en&formkey=dDlsbDlnclNrc3dIS2otRFdxUEFTNnc6MQ#gid=0 mnenje] najkasneje v treh dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO1 Povzetki seminarjev 2011

2011-05-29T08:13:17Z

MatjaZalar: /* Ula Štok: Mutacija mitohondrijske DNA v povezavi z rakom debelega črevesa kot posledica abnormalnega delovanja citokroma c oksidaze */

== Alja Zottel: Vloga imunskega sistema pri nastanku ateroskleroze ==
Glavni vzrok nastanka ateroskleroze je imunski odgovor na lipoproteine majhne gostote oz LDL, ki se kopiči pod endotelom arterijskih žil. Apolipoprotein B100, ki je komponenta LDL, se veže na proteoglikane zunajceličnega matriksa in se pod vplivom različnih radikalov oksidira. OxLDL nato aktivira endotelijske celice, da začnejo proizvajati adhezijske beljakovine, kot sta E-selektin in VCAM-1. Te beljakovine skupaj s kemokini povlečejo monocite, T limfocite in in dendritske celice v endotelijsko plast žile. Monociti se nato pod vplivom M-CSF citokina diferencirajo v makrofage. Makrofagi nato začnejo proizvajati odstranjevalne receptorje. Ti tako lahko prepoznajo oxLDL in ga z endocitozo vsrkajo. Makrofagi se zato napihnejo in spremenijo v »foam cell«. Te celice so najštevilčnejše celice v aterosklerotskih plakih. Dejavniki, ki pospešujejo nastanej ateroskleroze so signalni proteini PRR, T levkociti in proteini CRP. T celice pomagalke izločajo interferon gama, ki privlači monocite. Protein CRP se veže na navadni LDL in tako ga lahko makrofagi, ki imajo receptorje za CRP vsrkajo. Dejavniki, ki preprečujejo nastanek ateroskleroze so B limfociti in protein PPAR. PPAR je receptorski protein oz. transkripcijski faktor, ki preprečuje nastanek »foam cell« celic in vsrkavanje LDL v makrofage. Preprečuje tudi razvoj T celic in povečuje količino HDL v krvi.

== Veronika Jarc: Hepatitis C ==
Hepatitis C(HCV) je nalezljiva bolezen, ki napade ljudi, šimpanze ter nekatere majhne modelne živali. HCV spada med RNA viruse z ovojnico.Razvrščen pa je v rod hepacivirus ter družino flaviviridae. Sestavljen je iz 6 genotipov (1-6), ki se razlikujejo v nukleotidni sekvenci od 30-35%, sedmega pa so odkrili leta 2008 (Gottwein et al., 2008). HCV vsebuje pozitiven trak gena (9,6 kb), ki je sestavljen iz 5´-NCR( non-coding region), 3´- NCR in IRES( internal ribosome entry side). IRES vsebuje odprto bralno ogrodje, ki šifrira strukturne in ne strukturne proteine. Med strukturne proteine spadajo proteinsko jedro, virusna RNA ter dva glikoproteina E1 in E2. Sestavni deli ne strukturnih proteinov pa so hidrofoben protein p7, NS2-3 proteaza, NS3 serin proteaza, NS4A polipeptid, NS4B protein, NS5A protein in NS5B RNA odvisna RNA polimeraza (RdRp).
S pomočjo različnih odkritij, kot so HCVpp(sestavljen iz lipidne ovojnice z E1-E2 proteini, na retrovirusni nukleokapsidi), izoliranje kloniranega gena 2a ter s pomočjo tega gena HCVcc( cell-culture produced HCV), so znanstveniki začeli preučevati življenski cikel in celično strukturo hepatitisa C. To so dosegli z preučevanjem različnih eksperimentalnih modelov kot so imunski odzivi, NK celice in dendritske celice.
Poznamo tudi proteine, ki jih HCv sreča v hepatocitski celici in ti so in tegrin RGE/RGD, LDL receptor, HDL receptor, klaudin okludin in tetraspanin CD81.

== Matja Zalar: Protein p53 ==
Protein p53, včasih imenovan tudi varuh genoma, kodira gen TP53 na sedemnajstem kromosomu. Je eden izmed tako imenovanih tumor-supresorskih proteinov, ki, kot to sporoča že samo ime, zavirajo nastanek in rast tumorjev. Na področju razumevanja delovanja, vloge in strukture proteina p53 in njegovih mutantov se izvaja veliko raziskav. Trenutno je p53 najbolj raziskan tumor-supresorski protein, še zdaleč pa ni edini. Gre za protein, ki se kopiči v jedru in z vezavo na DNA v obliki teramera nadzoruje in regulira procese kot so apoptoza, zaustavitev celičnega cikla in popravljanje poškodovane DNA. Za raziskovalce je še posebno zanimiv zaradi dejstva, da v nemutirani obliki zavira nastanek in rast tumojev, njegove GOF mutirane oblike pa pripomorejo k nenadzorovani delitvi celic in nastanku rakastih tkiv. Veliko raziskav se ukvarjaja z iskanjem snovi, ki bi obnovile osnovno obliko p53, oziroma uničile mutantske oblike p53 v rakastih celicah ter s tem uničile tumor. To pa bi lahko bistveno izboljšalo tehnike zdravljenja rakavih obolenj in odziv človeškega organizma na ta zdravljenja. Odkrili so že kar nekaj takšnih snovi (RITA, PRIMA, nutlin3), ki pa jih še vedno testirajo in še niso v redni uporabi pri zdravljenju rakavih obolenj.

== Andrej Vrankar: Androgena alopecija ==
Na podlagi raziskav, ki so jih znanstveniki izvedli na celičnih vzorcih posameznikov z androgeno alopecijo, so ugotovili, da je bila domneva, da je za nastanek AGA kriv propad matičnih celic v lasnem mešičku oziroma, propad samega lasnega mešička napačna. Raziskave so pokazale ravno nasprotno in sicer, da se matične celice tudi v plešastem lasišču posameznika z AGA ohranjajo in da lasni mešički ne propadejo, vendar se le zelo skrčijo. So pa ugotovili, da se število celic imenovanih predniške celice v plešastem lasišču močno zmanjša, kar je eden od glavnih vzrokov za nastanek AGA, saj so prav predniške celice tiste, ki so zaslužene za rast las. Čeprav se dednost smatra kot glavni vzrok za nastanek AGA, pa tudi hormoni igrajo pomembno vlogo. Pri moških je to moški hormon testosteron, ki se s pomočjo encima 5-α-reduktaze v lasno mešičnih celicah pretvarja v svojo bolj aktivno obliko dihidrotestosteron (DHT). Ta se se nato s posebno vezjo veže na androgene receptorje v lasnih mešičkih, kar sproži posebne procese, ki skrajšajo anageno fazo celičnega cikla. Zaradi skrajšanja te faze las prej prestopi v telogeno fazo in izpade. Kako občutljivi so lasni mešički na androgene pa je seveda gensko pogojeno.

== Sandi Botonjić: Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom ==
Kisikovi radikali, ki povzročajo oksidativni stres lahko v skrajnem primeru poškodujejo DNA in tako povzročijo nenadzorovano delitev celic, kar pomeni nastanek raka v organizmu. Hkrati pa je raven kisikovih radikalov v rakastih celicah višja, kot v zdravih, in sicer zaradi onkogenih stimulacij, povečane presnovne aktivnosti ter okvare mitohondrijev. Toda rakave celice imajo, kot protiutež tudi močan antioksidantni mehanizem s katerim zavirajo programirano celično smrt.

Raziskovalci so tekom analiziranja večih tkiv, ki so obolela z različnimi vrstami raka ugotovili, da je pri vseh povečana raven [http://www.thesgc.org/structures/structure_images/2WZ9_400x400.png tioredoksinu podobnega proteina - TXNL2]. Zatem so izvajali poskuse na miših tako, da so jim vbrizgali kancerogene eritrocite in ko so se pojavili simptomi tumorja – so jim vbrizgali še protein TXNL2. Ugotovili so, da protein TXNL2 zavira rast rakavih celic. Proučevali so tudi vpliv proteina TXNL2 v mišjih zarodkih. Prišli so do zaključka, da protein TXNL2 regulira raven kisikovih radikalov tako pri živečih organizmih, kot med embriogenezo. Znanstveniki so prepričani, da je protein TXNL2 potencialna tarča bioloških zdravil v prihodnosti.

== Ana Dolinar: Prilagojena ali prilagodljiva imunost? Primer naravnih celic ubijalk ==
Naravne celice ubijalke (NK celice) so vrsta levkocitov. V človeškem telesu so zadolžene za uničevanje patogenih organizmov s pomočjo za celice strupenih snovi. Na površini imajo pet skupin receptorjev: aktivacijske, inhibitorne, kemotaksične in citokine ter adhezijske receptorje.

Njihova aktivacija je odvisna od vezave ligandov na površinske receptorje NK celice. Če je vezanih več inhibitornih ligandov kot aktivacijskih, potem se NK celica ne aktivira, ker inhibitorni ligandi zavrejo delovanje NK celice. V primeru, da se veže več aktivacijskih kot inhibitornih ligandov ali pa se slednji sploh ne vežejo, se NK celica aktivira ([http://www.georg-speyer-haus.de/agkoch/research/subframe_en.htm aktivirana NK celica-rumeno, tarčna celica-rdeče]). Vezava kemotaksičnih ligandov vpliva na gibanje molekule zaradi kemičnih signalov, vezava citokinov spodbuja rast celic ali sintezo snovi, ki jih potrebuje imunski sistem, vezava adhezijskih ligandov pa omogoča pritrjanje NK celice na tarčno celico.

Raziskovalci se trudijo, da bi našli optimalno imunoterapijo, pri kateri bi sodelovale NK celice. Te terapije bi bile uporabne predvsem pri rakavih obolenjih, vendar so možnosti tudi pri obolenjih z virusom HIV ali z virusom hepatitisa C. Ta način imunoterapije je mogoč, ker večina tumorskih celic in virusov ne izraža MHC tipa 1, pomembnega inhibitorskega liganda za NK celice. [http://media.wiley.com/CurrentProtocols/IM/ima01n/ima01n-fig-0004-1-full.gif Zgradba MHC-1 molekule, prikazana z Ribbonovim diagramom in vezanim peptidom (A) ter površinska struktura molekule z vezanim peptidom (C). Slika B prikazuje molekulo MHC-2 z vezanim peptidom.]

== Urška Rauter: Razvojne vloge Srf, kortikalnega citoskeleta in celične oblike pri orientaciji epidermalnega vretena ==
Mehanizem nastajanja polariziranega epidermalnega sloja, ki s procesoma stratifikacije in diferenciacije tvori kožo, regulira več različnih med seboj v komplekse povezanih bioloških molekul. Trije najbolj osnovni procesi so delovanje proteinov aktina, orientacija vretena in sistem celične signalizacije. Znanstveniki pa so v obširni raziskavi potrdili tudi pomembno vlogo t. i. Srf proteina (serum response factor protein), transkripcijskega dejavnika, katerega pomembna vloga je regulacija celične diferenciacije.

Srf je transkripcijski dejavnik, ki se veže na določen, njemu ustrezen receptorski element; Sre (serum response element), to so predvsem geni v zgodnjem razvoju, geni za razvoj nevronov in mišična gena (proteina) aktin in miozin. Ker je njegova primarna funkcija regulacija ekspresije naštetih genov, odločilno vpliva na celično rast in diferenciacijo, prenos med nevroni in razvoj mišic.

Namen raziskave je obširen. Rezultati obetajoči. Dokazali so pomembno vlogo Srf proteina pri marsikaterem mehanizmu/procesu v embrionalnem razvoju. Tako recimo Srf odločilno vpliva na diferenciacijo celic, saj izguba le-tega povzroči kaotično deljenje in diferenciacijo celic med več plastmi epidermisa. Nadalje vpliva tudi na pravilno vzpostavitev polarnosti bazalne lamine in še najbolj ključno na tvorbo aktinsko-miozinskega skeleta, ki je nujen za pravilno mitozo, posledično za obliko in trdnost celice. Orientacija vretena in asimetrično dedovanje sta po zadnjih raziskavah osrednja mehanizma, ki omogočata matičnim celicam samostojno obnovi in diferenciacijo v pravilni smeri. Rezultati kažejo, da lahko takšne signale pošiljamo preko Srf proteina in aktinsko-miozinskega skeleta, za pravilno tvorbo in nadzirano regulacijo orientacije vretena, asimetrične celične delitve in nasploh usodo posamezne celice. Rezultati razkrivajo nove pojasnitve bioloških procesov, ki sodelujejo pri tvorbi morfologije epidermisa.

== Špela Pohleven: Prioni ==

Prioni so patogeni proteini, ki se od svojih nepatogenih, normalnih, v zaporedju aminokislin enakih dvojnikov, razlikujejo v 3D strukturi – imajo večji del β ploskev. Poznamo več vrst prionov, toda običajno govorimo le o proteinu PrP, ki je prisoten pri ljudeh in živalih. Ostali so namreč značilni za glive, ki so tako primerne za razne raziskave.
Za prione je značilno povezovanje v nitaste polimere, ki jih imenjujemo amiloidi. Znanstveniki domnevajo, da je prav njihova urejena struktura tista, zaradi katere so slabo topni v detergentih in odporni na proteaze.
Najbolj nenavadna lastnost prionov pa je njihova zmožnost širjenja brez potrebe po DNA in RNA. V zvezi s tem potekajo številne raziskave, saj prioni povzročajo številne smrtne bolezni, kot so Creutzfeldt-Jakobova bolezen, smrtonosna družinska nespečnost in druge. Z informacijami, ki jih tako pridobivajo, je možnost za odkritje zdravila večja.
Pri eni od nedavnih raziskav so tako ugotovili, da obstajata dve prionski obliki proteina PrP – infektivna in toksična. Za raziskave so uporabili miši z različnim izražanjem gena PRNP za PrP protein. Vse so okužili s prioni praskavca (ena od prionskih bolezni). Vse so dosegle enak prag infektivnosti, toda smrt ni nastopila istočasno. Iz meritev so znanstveniki prišli do zaključka, da morata obstajati dve različni obliki. To pa je le izhodišče za nove raziskave.

== Maša Mohar: Sladkorna bolezen, kot bolezen imunskega sistema ==

Diabetes mellitus je kronična motnja metabolizma beljakovin, lipidov in ogljikovih hidratov. Nastane zaradi zmanjšane funkcije proizvajanja insulina v telesu. Njen vzrok pa je lahko studi zmanjšana sposobnost telesnih celic za pravilno izkoriščanje insulina. Tip 2 je od insulina neodvisen diabetes (NIDDM). Ta tip ima 80-90% vseh pacientov in se pojavi v odraslem obdobju življenja, spodbudijo ga lahko različni mehanizmi, in za nekatere se še ne ve točno kako pride do tega, je pa res da k temu veliko pripomore nezdrav način življenja in seveda dednost. Prav tako se diabetes tipa 2 deli v dve skupini in sicer na debeli tip, ki ga ima približno 80% vse populacije in na ne debeli tip.
Da je T2D bolezen imunskega sistema pa ugotovimo s tem ko vidimo kako se telo odzovena določene mehanizme, ki sprožijo to bolezen. To so oksidativni stres, stres ER( endoplazemski retikel), lipotoksičnost in glukotoksičnost. Prav tako je potrebno poudariti, da ima diabetes tipa 2 svoje metabolne karakteristike in skupaj s temi patogenimi mehanizmi tvori formulo za nastanek bolezni. Seveda lahko pri T2D pride tudi do dolgoročnih komplikacij, kot so makro in mikro- vaskularne bolezni, problemi z ledvicami, očmi in živci. Te pa so glavni dejavniki za povzročitev hujšega bolezenskega stanja in ne nazadnje tudi smrti zaradi diabetesa.

== Mirjam Kmetič: Regulacija celičnega metabolizma železa ==

Železo je pomemben mikroelement, ki ga vezanega na proteine, vsebujejo skoraj vsa živa bitja. Celice sesalcev potrebujejo zadostno količino železa, da zadovoljijo metabolne potrebe ali dosežejo specializirane funkcije. Vsekakor pa je železo potencialno strupeno, še posebej v obliki Fe2+ ionov, ki katalizirajo pretvorbo vodikovega peroksida v proste radikale, ti pa poškodujejo veliko celičnih struktur (DNA, proteine, lipide...) in posledično celica lahko celo odmre. Vse oblike življenja se temu izognejo tako, da vežejo železove ione na proteine in tako hkrati izkoristijo njegove ugodnosti. Železo se prenaša v tkivo ob pomoči kroženja transferina, prenašalca, ki veže železo v plazmi, katerega predvsem sproščajo črevesne resice in retikuloendotelni makrofagi. Z železom bogat transferin se veže na membranski transferin receptor 1, kar se odraža z endocitozo in sprejemom te kovine. Sprejeto železo se prenese do mitohondrija za sintezo hema ali železo-žveplovih proteinov, ki so bistveni deli mnogih metaloproteinov. Presežno železo se skladišči in detoksificira v feritinu, ki je v citosolu. Metabolizem železa je nadzorovan na različnih nivojih in z raznovrstnimi mehanizmi. Pri uravnavanju je zelo pomemben sistem IRE (iron-responsive element)/IRP (iron-regulatory protein), dobro poznano post-transkripcijsko regulatorno vezje, ki ne le vzdržuje homeostazo v različnih tipih celic, ampak tudi prispeva k sistemskemu ravnovesju železa.

== Lea Kepic: Agonisti adrenoreceptorjev β2 ==

Vloga receptorjev v organizmih je zelo pomembna saj prenaša vse potrebne informacije za delovanje. Delimo jih na ionotropne in metabotropne. Največja skupina metabotropnih receptorjev pripada receptorjem, ki so sklopljeni s proteinom G. Mednje spadajo tudi adrenergični receptorji ali adrenoreceptorji. Adrenoreceptorji so tarčni za katekolamine (fight or flight hormoni) med katere spadajo adrenalin, noradrenalin in dopamin. V svojem seminarju sem se posvetila predvsem podskupini β2 (β2-AR) in njihovim agonistom. Agonisti so spojine, ki se selektivno vežejo na specifične receptorje, ki sprožijo nadaljnji odziv. Njegova naloga je posnemanje naravno obstoječih (endogenih) molekul, kot so na primer hormoni. Najbolj pogost in učinkovit agonist za β2-AR je izoprenalin, med hormoni pa je najboljši adrenalin. S pomočjo eksperimentov znanstveniki raziskujejo posebnosti v zgradbi predvsem kristalnih struktur, tvorbo vezi z različnimi spojinami, konformacijske spremembe, vpliv inhibitorjev, ravnotežna stanja ter energijska pretvarjanja. Rezultati teh raziskav so izhodišče za praktično uporabnost agonistov. Zaradi njihovih lastnosti jih vedno več uporabljamo v medicini za zdravnjenje plujčnih bolezni; predvsem astme in bronhitisa. To področje za enkrat še ni do dobra raziskano zato jih navadno uporabljamo le kot dodatke drugim zdravilom. Raziskani pa so že tudi nekateri negativni učinki na telo.

== Iza Ogris: Zakaj imajo možgani glikogen? ==

Glikogen se v možganih nahaja v precej manjših koncentracijah kot v jetrih in mišicah.Pojavi se vprašanje o njegovi vlogi v možganih in kje se nahaja. Glikogen vsebujejo astrocite- glia celice, ki obdajajo nevrone in skbijo za koncentracijo ionov v izvenceličnem prostoru ter dovajanje določenih snovi nevronom. Ko se med aktivnostjo nevronov v izvenceličnem prostoru kopičijo kalijevi ioni, jih astrocite začnejo privzemati z K/Na ATPazo. Posledično se v astrocitah zviša nivo AMP, kar stimulira delovanje encima glikogen fosforilaze (razgradnja glikogena). Astrocite med nevronsko aktivnostjo privzemajo tudi živnčni prenašalec glutamat iz sinaps, ki tudi posredno povzroča padec energije v astrocitah. Ko se nivo glukoze v dejavnih nevronih znižuje, se medtem v astrocitih povečuje. Koncentracija glukoze je nato v astrocitih večja kot v izvencelični tekočini in nevronih, zato se ustvari koncentracijski gradient kar omogoči pot glukoze iz astrocitov v nevrone. Pri vzdrževanju glukoze se tako razgradnja glikogena izkaže za bolj učinkovito kot le privzem glukoze iz krvi. Razkriva se izvor in usoda glukozne rezerve.

== Ines Kerin: Kanabinoidi za zdravljenje shizofrenije? Uravnotežena nevrokemična sestava za škodljive in terapevtske učinke uživanja konoplje ==

Že desetletja velja prepričanje, da je uživanje konoplje eden pomembnih dejavnikov za nastanek in razvoj shizofrenije. Vendar so v novejših raziskavah odkrili, da naj bi kanabinoidi, psihoaktivne substance v konoplji, izboljšali nevropsihološke učinke in negativne simptome, ter imeli antipsihotične lastnosti pri ljudeh s shizofrenijo. Shizofrenija je huda duševna bolezen iz skupine psihoz. Simptome shizofrenije povzroča spremenjena količina določenih snovi v možganih, in sicer živčnih prenašalcev, ki omogočajo medsebojno komunikacijo možganskih celic. Motnje v komunikaciji pa povzročajo spremembo v delovanju možganov. Pomembno vlogo ima pri bolezni dopamin, ki lahko s prevelikim sproščanjem izzove nekatere simptome.
Shizofrenijo zdravijo s pomočjo antipsihotikov, ki imajo podobne lastnosti kot kanabinoidi v konoplji. Vendar se učinki konoplje od učinkov antipsihotikov nekoliko razlikujejo. Pri negativnih simptomih konoplja, tako kot antipsihotiki, spodbuja sproščanje in delovanje dopamina. Manj znano pa je, ali zavira ali spodbuja delovanje ostalih petih nevrotransmiterjev (serotonina, acetilholina, noradrenalina, glutamina in GABA). Na pozitivne simptome ima konoplja, kot je vidno v tabeli lahko tako koristne kot nekosristne učinke. Simptome lahko izboljša z zaviranjem sproščanja serotonina, acetilholina in noradrenalina. V primeru dopamina, glutamata in GABA ima konoplja negative učinke, saj v nasprotju z antipsihotiki, poveča sproščanje dopamina in zavira delovanje glutamata in GABA. Obstajajo dokazi, da imajo kanabinoidi zdravilne učinke na pozitivne in negativne simptome pri shizofreniji. Vendar to poglavje še ni zaključeno in se izvajajo še nadalnje raziskave v tej smeri.

== Eva Knapič: Kako virusi vodijo delovanje celice. ==
Virusi so geni obdani z zaščitno proteinsko ovojnico. Za izražanje teh genov, da lahko naredijo proteine in podvojijo kromosome, je potrebno, da vstopijo v celico in uporabijo celične mehanizme, saj sami tega niso zmožni. Poznamo več vrst virusov. Posebnost evkariontskih virusov je sposobnost posnemanja kratkih linearnih motivov proteinov poznanih pod kratico SLiMs. To so deli proteinov, ki so odgovorni za posredovanje med nekaterimi celičnimi funkcijami. So zelo kratki, večinoma nekje od 3 do 10 aminokislin. Motivi sodelujejo pri vezavi proteinih, pri prepoznavanju post-translacijske modifikacije encimov, pri usmerjanju proteinov v celične razdelke in pa so prisotni na cepitvenih mestih proteina. S posnemanjem različnih motivov lahko virusi prevzamejo nadzor nad celico. Najpogostejši mehanizmi prevzema nadzora so uporaba celičnega transporta, manipuliranje signalnega transporta, nadzor proteinov v celici, regulacija prepisovanja, sprememba modifikacije gostiteljevega proteina in usmerjanje modifikacije proteinov.
Uporaba proteinskih motivov v celici in lahko posnemanje le teh predstavlja šibkost v celični organiziranosti, saj virusi s pridom izkoriščajo to v svojo korist. Posnemanje motivov virusom omogoča, da sami vodijo delovanje celice in se sami s pomočjo celičnih mehanizmov enostavno razmnožujejo in tako hitro okužijo celoten organizem.
V nadalje bodo potekale raziskave za izkoriščanje posnemanja motivov v namene zdravljenja virusnih okužb.
== Katra Koman: Pomen dendritskih celic (DCs) in celic ubijalk (NK) v imunskem odzivu na okužbo z virusom HIV-1 ==
Dendritske celice (DCs - dendritic cells) in celice ubijalke (NK – natural killer cells) sta dva tipa celic prirojenega imunskega sistema, ki imata zelo pomembno vlogo pri protivirusni odpornosti. Tako dendritske celice, kot tudi celice ubijalke so sicer pomemben del (nespecifičnega) prirojenega imunskega sistema, a hkrati vplivajo tudi na učinkovit razvoj (specifičnega) prilagojenega imunskega odziva. DC so ključnega pomena za aktiviranje za virus specifičnih T celic, kar pa je močno odvisno od prejšnjega, prirojenega imunskega odziva. NK celice pa ovirajo zgodnje širjenje virusov, tako da proizvajajo citokine in s fagocitozo neposredno uničujejo okužene celice. Razumevanje delovanja in funkcije teh celic pa ima pomemben vpliv na razvijanje nove strategije cepiva proti virusu HIV-1, katere uspeh bo odvisen od primernega razumevanja delovanja teh celic.

== Jana Verbančič: Apoptozi podobna smrt v bakterijah, ki jo povzroča HAMLET, lipidno-proteinski kompleks v človeškem mleku ==
Apoptoza oz. programirana celična smrt je eden najpomembnejših procesov v evkariontskih celicah. Organizem je z apoptozo sposoben sam uravnavati število živih celic. Uniči jih lahko, ker so poškodovane, stare ali ker ne opravljajo več svoje naloge, lahko pa uniči tudi popolnoma zdrave celice, ki jih ne potrebuje več (npr. pri embrionalnem razvoju). Pomemben dejavnik pri apoptozi so encimi kaspaze, ki cepijo in aktivirajo druge proteine, vse skupaj pa lahko poteka po dveh poteh. Prva je notranja in vključuje mitohondrije in citokrom c, ki deluje kot signalna molekula v apoptotskem ciklu ter tako sproži delovanje kaspaz in posledično apoptozo. Druga pot je zunanja in vključuje aktivacijo proteinskih receptorjev (t. i. receptorjev smrti) na zunanji strani membrane. Oblikuje se kompleks iz receptorja, adaptorskega proteina in vezane kaspaze (DISC), ki povzroča cepljenje in aktivacijo nadaljnjih kaspaz; to pa spet vodi v apoptozo. V mehanizme so lahko vključeni mnogi drugi proteini ali neproteinski signali.
Programirane celične smrti pa nimajo samo evkarionti, ampak so dokazali, da so tudi prokarionti sposobni procesov, ki so zelo podobni apoptozi. Raziskave so delali na streptokokih in tumorskih celicah, ki so jim dodali kompleks HAMLET (human alpha-lactalbumin made lethal to tumor cells), ki ga lahko najdemo v človeškem mleku. Kompleks je deloval kot signalna molekula za začetek apoptoze v tumorskih celicah oz. za začetek apoptozi podobnega procesa v bakterijah.

== Ana Remžgar: Črevesna absorpcija vitamina D ne poteka le s pasivno difuzijo: dokazi za vpletenost enakih transporterjev kot pri holesterolu ==
Vitamin D je hormon, ki ga telo lahko proizvede samo s pomočjo obsevanja kože z ultravijolično svetlobo, vendar je hipovitaminoza D razširjena v mnogih državah in je pomemben svetovni zdravstveni problem. Vitamin D je nujno potreben za uravnavanje ravnovesja med kalcijem in fosfati v telesu ter za normalno rast kosti.

Dolgo časa je veljalo, da se v črevesju vitamin D absorbira le s pomočjo pasivne difuzije. Znanstveniki so kulturi človeških embrionalnih ledvičnih (HEK) celic dodali vsaj enega od teh membranskih proteinov (SR-BI, CD36, NPC1L1). Ti trije proteini so pomembni pri absorpciji holesterola. Zaradi podobne zgradbe holesterola in vitamina D, so znanstveniki sklepali, da so lahko ti trije proteini pomembni tudi pri absorpciji vitamina D. Ko so HEK celicam dodali te proteine, se je absorpcija vitamina D opazno povečala. Ko pa so HEK celicam dodali poleg proteinov še njihove inhibitorje, se je absorpcija močno zmanjšala.
Vpliv SR-BI so opazovali tudi in vivo. Uporabili so wild type miši ter miši z mnogo bolj izraženim Scavenger receptorjem razreda B tipa I (SR-BI). Tudi tu se je pri SR-BI miših povečala absorpcija vitamina D.

Ti rezultati nam prvič pokažejo, da se vitamin D v črevesju ne absorbira le preko pasivne difuzije vendar je v ta proces vključenih kar nekaj transporterjev.

== Maja Grdadolnik: Jedrni in nejedrni receptorji za estrogene. ==
Receptorji za estrogene oz. estrogenski receptorji so proteinske molekule z vlogo specifičnega mesta vezave ustreznega liganda. Nahajajo se v vseh celicah tkiv, ki so tarčne celice estrogena. Lahko se nahajajo v jedru celice, v neposredni bližini DNA, lahko pa so vezani na posebna mesta na membrani celice, t.i. caveole.
Estrogeni (estron (E1), estradiol (E2), estriol (E3)) so lipidopolarni in brez večjih težav prehajajo skozi lipidni dvosloj. Nato se vežejo na lipoproteine v krvi, ki jih prenesejo do jedra tarčne celice. Tarčne celice so po navadi celice jajčnikov, testisov, nadledvičnih žlez, jeter in prsi. V jedru se nato vežejo na estrogenski receptor, s katerim tvorijo kompleks. Ta ligand-receptor kompleks se nato s posebnim mestom (domeno E) veže na specifično mesto na DNA, imenovano estrogen response element (ESE). S tem sodeluje pri procesu transkripcije in uravnava sintezo ustreznih proteinov.
Nejedrni estrogenski receptorji so vezani na posebna mesta na membrani celice, t.i. caveole. Na ta mesta so vezani z integralnim proteinom, za vezavo pa potrebujejo aminokislinski substrat. Receptorje na membrani lahko povezujemo z interakcijo z različnimi ligandi, imajo pa tudi pomembno vlogo posredne aktivacije endotelijske NO sintaze, ki pozitivno vpliva na srce in ožilje. Nejedrne estrogenske receptorje že povezujejo s procesi, ki blagodejno vplivajo na kardiovaskularne bolezni in tkivo endotelija.

== Andreja Bratovš: Vloga GPCR v patologiji Alzheimerjeve bolezni. ==
Alzheimerjeva bolezen je najpogostejša oblika demence. Zaradi odmiranja nevronov pride do zmanjšanja obsega možganov in pešanja razumskih funkcij. Eden glavnih razlogov za nastanek bolezni so amiloidni plaki. Ti nastajajo s kopičenjem amiloidnih peptidov beta. APP (amyloid precursor protein) je membranski protein, ki ga pri zdravem človeku cepi najprej α-sekretaza (nastane sAPPα), nato pa še γ-sekretaza – nastane topen delec p3. Kadar pa APP cepi β-sekretaza, nastane najprej sAPPβ, po cepitvi z γ-sekretazo pa nastane amiloidni peptid beta.
Pri iskanju zdravila za Alzheimerjevo bolezen se trenutno osredotočajo prav na amiloidne plake oz. na preprečevanje njihovega nastajanja ter njihovo razgradnjo. Alternativen pristop imunoterapiji je regulacija receptorjev, sklopljenih z G-proteini, saj so ti udeleženi v več fazah nastajanja plakov. Možnih je več poti, in sicer: zaviranje nastajanja amiloidnih peptidov beta z regulacijo α-, β- ali γ-sekretaze ter sproščanje encimov za razgradnjo plakov. Pri regulaciji α-sekretaze gre za promoviranje njenega delovanja, saj se tako poleg tega, da ne nastajajo amiloidni peptidi beta, tudi sprošča sAPPα, ki ima vlogo pri zaščiti nevronov. Za β-sekretazo je sicer znanih veliko inhibitorjev, vendar jih iz možganov eksportira P-glikoprotein. Problem pri γ-sekretaze je, da ta sekretaza cepi tudi del proteina Notch, zato bi z njeno inhibicijo vplivali tudi na Notch signalno pot.

== Kaja Javoršek: Potencial matičnih celic pri Parkinsonovi bolezni in molekularni faktorji za tvorbo dopaminskih nevronov. ==
Parkinsonova bolezen je nevrodegenerativna bolezen bazalnih ganglijev. ta bolezen prizadane predvsem telesno gibanje, nastane pa ker se zmanjša koncentracija dopamina v striatumu. Kot posledica tega, začnejo propadati dopaminski nevroni v substanti nigri. Prav propadanje dopaminskih nevronov pa je vrzok za Parkinsonovo bolezen. Vzrok za propadanje teh nevronov pa še vedno ni znan. Znano je da dopaminski nevroni s starostjo pospešeno propadajo. To je tudi razlog, zakaj se ta bolezen pojavlja šele pri starejših ljudeh.
Danes se v medicini uporablja veliko terapij, ki pa le lajšajo simptome in bolezni ne pozdravijo. Prav to je razlog za tako veliko število raziskav povezanih s Parkinsonovo boleznijo. Čeprav mehanizmi razvoja dopaminskih nevronov še niso povsem znani, so raziskovalci odkrili kar nekaj molekularnih faktorjev, ki vplivajo na njihovo tvorbo, na primer Fox proteini in receptor sirota Nurr1. Fox proteini so transkripcijski faktorji, ki vežejo DNA. Med temi proteini igrata najpomembnejšo vlogo v nastanku dopaminskih nevronov FoxA1 in FoxA2 proteina. Receptor sirota Nurr1 pa je pomemben pri nastanku L-DOPE, ki je vmesen produkt pri nastanku dopamina iz L-tirozina. Za nastanek L-DOPE mora biti prisoten encim tirozin hidroksilaza. Za izražanje tega encima pa je pomemben receptor sirota Nurr1 in mutacije tega receptorja so povezane s Parkinsonovo boleznijo in shizofrenijo.
Poleg vseh načinov zdravljenja, pa poskušajo Parkinsonovo bolezen pozdraviti tudi s pomočjo matičnih celic, saj so se zmožne diferencirati v katero koli vrsto celic, vključno z dopaminskimi nevroni.

== Tamara Marić: Organizacija jedra. ==
Organizacija genoma v jedru je zelo kompleksna in dinamična in prav to je znanstvenike privedlo do mišljenja, da ima jedro neko globjo strukturo, kjer mora vladati red. S pomočjo novih tehnoloških metod (3C, FISH, 4C) so odkrili kar nekaj zanimivh stvari o sestavi samega jedra. Jedro si moramo predstavljati kot 3D strukturo, v kateri se neprestano nekaj dogaja. Sestavljen je iz dveh glavnih domen, obrobja in centra. Na obrobju sta še dva pododdelka. Ob jedrnih porah se nahajajo aktivni geni, ki so povezani s številnimi proteini, speči geni pa se nahajajo ob lamini. V centru se pododdelki medseboj razlikujejo po funkcijah. V jedrcu se nahajajo geni za rRNA, v transkripcijskih tovarnah se nahajajo vse »sestavine«, ki jih geni potrebujejo za prepis, polycombska telesca imajo bistveno vlogo pri ohranjevanju represije in perinuklearni prostor je specializiran za replikacijo heterokromatina. Ker pa to ne miruje je logično da kromatinske zanke med seboj interagirajo. Poznamo homologne (kjer gre za podobno zgrajene/iste kromosome) in nehomologne(se med seboj razlikujejo) interkromosomske interakcije. Pri prvi je pomembno, da se podobna kromosoma »zmenita«, kateri bo aktiven, pri drugi pa je diferenciacija celice odvisna od aktivnosti nekega gena.

== Mitja Crček: Matične celice in njihova vloga pri zdravljenju bolezni in poškodb. ==
Regeneracija je proces, pri katerem nadomestimo poškodovane telesne dele. V človeškem telesu imajo to nalogo matične celice (MC), ki skribijo za delno regeneracijo in celjenje poškodb. Matične celice so nediferencirane celice odraslege človeka ali zarodka, ki imajo izjemen potencial, da se defirencirajo v mnogo različnih tipov celic v telesu. V tri do pet dni starih zarodkih iz matičnih celic nastane celotno telo organizma, pri odraslih ljudeh pa nas matične celice ohranjajo pri življenju. Glede na potentnost jih razdelimo v štiri razrede: totipotentne in pluripotentne MC so celice, ki se lahko diferencirajo v praktično vse celice telesa, medtem ko so multipotentne in unipotentne bolj omejene. Drugo delitev lahko opravimo glede na njihov izvor: embrionalne MC izvirajo iz zarodka, medtem ko MC odraslih tkiv in organov najdemo med že diferenciranimi celicami. Zaradi vseh njihovih lastnosti imajo velik potencial pri zdravljenju bolezni, že vrsto let jih uporabljajo za zdravljenje levkemije in limfoma. Z diferenciacijo MC v nevrone bi lahko pozdravili poškodbe hrbtenjače in možganov, ob sproščanju kemičnih signalov iz MC proti lasnim mešičkom bi lahko pozdravili plešavost. V teoriji bi lahko nadomestili tudi izgubljen zob, zdravili slabovidnost in gluhost, pa tudi sladkorno bolezen in neplodnost. Velik potencial imajo tudi pri zdravljenju poškodb kosti in mišic. Pri zlomih ter poškodbah hrustanca in vezi služijo predvsem za hitrejšo regeneracijo, omogočajo pa tudi zdravljenje mišične distrofije ali pa povečanje mišične mase in moči, kar bi lahko s pridom izkoriščali športniki in starejši ljudje.

== Sara Lorbek: Sovplivanje maščobnih kislin ter genov na adipokine in debelost. ==
Belo maščobno tkivo ni namenjeno zgolj shranjevanju zalog maščobe, temveč ima velik vpliv na prisotnost in stopnjo vnetja v telesu, saj na le-to vpliva s sekrecijo adipokinov. Adipokini so proteini, ki se izločajo iz celic maščobnega tkiva, do danes pa je poznanih že več kot 100 različnih. Njihov vpliv je zelo različen, v nalogi pa sem se osredotočila na vpliv aipokinov na vnetje, za katerega velja, da ga sproža debelost. Adipokina, ki odločilno prispevata k vnetnemu stanju sta TNF in interlevkin-6 (IL-6), njuna količina pa je močno odvisna tudi od telesne teže: večja kot je telesna teža posameznika, več je teh dveh adipokinov, ki promovirata vnetje, zato je tudi stopnja vnetja večja pri debelejših osebkih. Maščobne kisline veljajo za snovi, ki so sposobne regulirati proizvodnjo adipokinov in s tem vplivati na stopnjo vnetja, toda natančni molekulski mehanizmi tovrstne aktivnosti maščobnih kislin še niso pojasnjeni. Kljub temu imamo že dovolj dokazov, da lahko z gotovostjo trdimo, da različni tipi maščobnih kislin različno ugodno/neugodno vplivajo na promocijo vnetja v organizmu, tako npr. uživanje večkrat nenasičenih in omega-3 m.k. znižuje količino IL-6 in TNF- torej zavira vnetje, uživanje nasičenih m.k. pa vnetje promovira, saj zvišuje količino IL-6 in TNF v organizmu. Odziv številnih adipokinov na različne m.k. do danes še ni bil raziskan, kar predstavlja nov izziv za področje nutrigenomike.

== Maja Remškar: Evolucijska dinamika transponibilnih elementov v majhnem RNA svetu ==
Genom si pogosto predstavljamo kot nekaj statičnega, a ni tako. V zadnjem času so odkrili transponibilne elemente, samostojne dele DNA, ki se lahko premeščajo po genomu in sprožajo mutacije. Če se vgradijo v v stukturne gene, običajno uničijo njihovo informacijo, če pa se vgradijo v regulacijske regije vplivajo na izražanje genov, navadno jih naredijo neaktivne. Vsebujejo gene za podvajanje in premeščanje. Sestojijo iz obrnjenih ponovitev na vsakem koncu in iz vsaj še gena za transpozazo, ki mu omogoča premeščanje. Vmes imajo lahko poljubno število genov. Transpozoni naj bi bili odvečna in sebična DNA in dolgo je bila naravna selekcija edini poznan pojav, ki je nadzoroval njihovo pretirano razmnoževanje. Dandanes vemo, da je mehanizmov njihovega zaviranja več, in sicer, lahko delujejo samorepresorsko (za vrste, ki se razmnožujejo nespolno), pri zaviranju lahko pomaga represorski alel, ki je navadno s škodljivimi TE v stiku, lahko pa jih nadzorujeta mehanizma siRNA in piRNA, ki vztrajno popravljata napake povzročene s strani transpozonov. Na dinamiko represorskih alelov vpliva genetski zdrs – zaradi zmanjšanja populacije, se poruši naravno ravnovesje in lahko pride do fiksacije škodljivejših alelov – in rekombinacija, ki prekine povezave med represorskimi aleli in njihovim tarčnim mestom vezave ter prepreči njihovo fiksacijo. Transpozone so preučevali na koruzi, v bakterijah, vinski mušici in tudi pri človeku, saj povzročajo dedne bolezni kot sta hemofiliji A in B ter Duchennova mišična distrofija.

== Rok Štemberger: virus HIv in povečana ekspresija ter imunogenost HIV-1 proteaze po deaktivacija encimske aktivnosti ==
Virus HIV spada v skupino retrovirusov in je povzročitelj ene najhujših svetovnih pandemij, ki vsako leto terja skoraj 3 milijone žrtev. Virus HIV potrebuje za svoje razmnoževanje gostiteljsko celico, ker nima svojih lastnih mehanizmov, s katerimi bi se lahko razmnoževal. Njegov razmnoževalni cikel obsega veliko procesov, ki se morajo izvršiti, da se virus HIV lahko ustrezno replicira. V moji raziskavi je bil pod drobnogled vzet eden izmed treh encimov, ki sodelujejo pri razmnoževanju HIV-a, in sicer HIV-1 proteaza. HIV-1 proteaza je encim, ki dolge verige proteinov cepi na manjše dele. Če se HIV-1 proteazo z inhibitorji blokira, bi to pomenilo da virus HIV ne bi imel potrebnih encimov za svoje razmnoževanje, saj je dolga veriga proteinov popolnoma neuporabna, če niso razrezani na manjše dele. V raziskavi so ugotovili, da če uporabimo mutirano HIV-1 proteazo, se ji aktivnost drastično zmanjša po drugi strani pa so opazili veliko ekspresijo. Ta ekspresija se je pokazal v tem, da je inducirala imunski odziv in HIV-1 proteaza je bila trača predvsem CD8+ T celic pomagalk. Kasneje so ugotovili, da lahko prav te T celice popolnoma uničijo mutirano HIV-1 proteazo iz telesa in jo s tem odstranijo iz našega sistema. Poskusi so bili narejeni tudi na transgenih miših, ki dajejo bolj verodostojne rezultate kot ostale miši. Ta raziskava bo osnova vsem nadaljnjim raziskavam, ki se bodo ukvarjali predvsem z HIV-1 proteazo, saj ta do sedaj ni bila deležna velike pozornosti. Različni Inhibitorji HIV-1 proteaze pa bodo v prihodnosti bili še bolj pogosto uporabljeni v mešanici zdravil proti bolezni HIV.

== Rok Vene: Spremembe nivoja metilacije DNA so sorazmerne s starostjo človeških možganov ==
V zaporedju DNA se nahajajo posebna zaporedja nukleotidov, ki so edina, na katerih lahko poteče metilacija. Ta mesta sestavlja dinukleotid CpG – cytosine-phosphat-guanine (Od 5' konca DNA verige proti 3' koncu sta citozin in gvanin zaporedno vezana s fosfodiestersko vezjo – 5'-CG-3'). Metilacija DNA je proces v katerem se na ta posebna t.i. CpG mesta veže metilna skupina (-CH3). Taka mesta so v DNA redkejša, kot bi statistično gledano smela biti. Večina CpG mest je metiliranih. CpG mesta se lokacijsko na DNA lahko nahajajo v skupkih imenovanih CpG otočki (CpG islands), ali posamič. Direktna posledica metiliranih CpG mest je utišanje genov, na katerih se ta metilirana mesta nahajajo. Skupaj z ostalimi epigenetskimi faktorji pa indirektno vplivajo še na diferenciacijo celic. Destabilizacija epigenetskih faktorjev je lahko vzrok za številne bolezni (rak, sindromi Rett, ICF, Prader-Willi,...).

V raziskavi iz članka so znanstveniki primerjali količino in lokacijo metilirane DNA v različnih možganskih tkivih. Precejšen del rezultatov je specifičen glede na vrsto tkiva (preko petsto lokusov), vendar obstajajo določene povezave, ki so značilne za vsa raziskovana tkiva možganov. Odkrili so deset specifičnih lokusov, ki vsi vsebujejo metilirana CpG mesta. Odkrili so tudi, da nivo metilirane DNA s starostjo tkiva narašča. Starost tkiva je bila v kar 32-75% primerov glavni razlog za spremembe v količini metilirane DNA. Nekatere izmed destih lokusov so že pred to raziskavo povezovali z starostnimi spremembami v metilaciji DNA, vendar so šest izmed desetih najpomembnejših lokusov odkrili na novo.

== Karmen Hrovat: Ciljanje kemokinih receptorjev v alergijskih boleznih ==
Kemokini so družina majhnih proteinov, velikosti 8-10 kDa. Sodelujejo v procesu agiogeneze, embriogeneze, za nas pa je bistveno, da spodbujajo premikanje levkocitov, bazofilcev, monocitov, jih usmerjajo in nadzorujejo njihov prehod iz krvi v tkiva. Dandanes jih uvrščamo med mnoge raziskave, povezujemo jih tako z aterosklerozo, prenosljivimi boleznimi kot sta virus HIV in malarija, rakom, luskavico in alergijskimi boleznimi med katere sodijo astma, alergijski rinitis in atopijski dermatitis. Poznamo štiri vrste kemokinov: CC,CCX, C in CX3C. Nekateri kemokini se vežejo na več različnih receptorjev in obratno.V članku je opisanih več poskusov na kemokinih receptorjih v miših obolelih za alergijskimi boleznimi.

Farmacevti si prizadevajo odkriti kemokine receptorje antagoniste, saj se je aktivacija GPCR kompleksa izkazala uporabna za zdravilo. V miših obolelih za alergijskimi boleznimi je bilo do sedaj tako in vitro kot tudi in vivo dokazanih že veliko antagonistov kemokinih receptorjev. Kljub temu številni zaradi vprašanja varnosti in farmacevtskih dogm niso dočakali kliničnega sojenja.Lahko rečemo, da napredek ovira tudi pomanjkljivo razumevanje funkcije kemokinov in njihovih receptorjev v alergijskih boleznih. Kljub temu pa se bodo v prihodnost namenili k iskanju antagonistov potencialnega kandidata kemokinega receptorja CCR3.

== Matevž Ambrožič: Termogene snovi in regulacija telesne teže ==
Eden izmed glavnih problemov modernega človeka je prekomerna teža in z njo povezane zdravstvene težave. Strokovnjaki so si edini, da je ključ do uspeha pozitivno razmerje med porabljeno in vnešeno energijo. Veliko pomoč pri porabi energije nudijo snovi, ki spodbujajo termogenezo, po možnosti prek oksidacije maščob. Katehini in kofein so termogene snovi, ki jih najdemo v mnogih naravnih virih, vsi skupaj pa se pojavljajo v čaju. Najboljša vira sta zeleni in beli čaj, saj sta manj obdelana. Pri izgubi telesne teže je vedno cilj večja poraba energije in uničevanje maščobnih zalog. Maščobe, ki jih zaužijemo, so v obliki triacilglicerolov in se shranjujejo v belem in rjavem maščobnem tkivu. Služijo nam kot rezervna zaloga energije, mehanska zaščita in pomoč pri vzdrževanju temperature. V določenih pogojih simpatični živčni sistem z izločanjem hormona epinefrina sproži pretvarjanje shranjenih triacilglicerolov v maščobne kisline (proces lipolize), te pa se lahko v procesu oksidacije porabijo za sintezo ATP. Da pospešimo porabo maščob, se s termogenimi snovmi skušamo vmešavati v metabolizem maščob na raznih stopnjah. Katehini in kofein katalizirajo lipolizo na različne načine, večinoma z inhibicijo zaviralcev lipolize. Rezultati raziskav sicer rahlo variirajo, vendar v splošnem velja, da katehini in kofein pomagajo pri regulaciji telesne teže.

== Marko Radojković: Vpliv rakastih celic in sepse na izraženost krvnega proteina trombina ==
Nedavne študije so pokazale kako anti-koagulanti pomagajo pri zdravljenju in preprečevanju raka, vendar natančen mehanizem ki opisuje kako sta strjevanje krvi in napredovanje raka povezana, ni bil znan do sedaj. Znastveniki so odkrili kako celice pod stresom povečajo proizvodnjo enega izmed ključnih faktorjev strjevanja krvi – trombina. Količina trombina ki ga naše celice proizvajajo je kontrolirana z dvema vrstami proteinov : proteini ki zavirajo produkcijo (FBP2 in FBP3) , ter proteini ki jo pospešujejo (hnRNPI, U2AF65 in U2AF35). Obe vrsti proteinov delujeta tako da se vežeta na celične ''stroje'' na protrombinski mRNA , in v normalnih pogojih, proteini inhibitorji vzdržujejo nizko koncentracijo trombina. Ko naše celice pridejo v stanje stresa, v primeru ko je povzročitelj vnetje, en drugi protein ki se imenuje p38 MAPK, reagira tako da pripne fosfatne skupine ihibitornim proteinom. To povzroči da se le ti težje vežejo na celične ''stroje'' za produkcijo trombina, in omogoča stimulatornim proteinom da prevzamejo glavno vlogo v mehanizmu. Torej, vnetje zaradi raka bi lahko pripeljalo do povečane ravni trombina in, kot je trombin glavni agent strjevanja krvi, bi to lahko pojasnilo, zakaj se pri bolnikih z rakom pogosteje pojavljajo krvni strdki.
Znastveniki so ugotovili da p38 MAPK protein tudi vpliva na proizvodnjo trombina v sepsi. Znana tudi kot zastrupitev krvi, sepsa se pojavi, ko bakterije ali drugi povzročitelji bolezni pridejo v kri, ki vodi k razširjenosti okužbe in nastanku težav pri strjevanu krvi. Ko so analizirali vzorce jeter odvzetih iz miši s sepso in iz bolnikov z rakom, so znanstveniki odkrili da je povečana produkcija trombina odgovor, tako kot na široko vnetje v primeru sepse, kot na lokalizirano vnetje v invaziji tumorja , kje se rakave celice širijo v bližnje tkivo.

== Urška Rode: Vpliv C- reaktivnega proteina na patogenezo simptomov metaboličnega sindroma ==
C-reaktivni protein je protein, akutne faze, ki nastaja v jetrih, njegova koncentracija v krvi se poviša ob razih vnetjih in okužbah, kot del imunskega odziva. Njegova vloga in pomen še nista povsem jasna. Najbolj znana njegova vloga je pri vezavi na fosfoholin na membrano bakterije ali poškovodovane celice. s tem ko se veže z eno strenjo na fosfoholin se na njegovo drugo stran veže prva komponenta klasične proti imunskega odziva.
njegova vloga na cel organizem še ni popolnoma pojasnjena. V zadnjem času poteka veliko raziskav, ki poučujejo njegov vpliv na sindrome metaboličnega sindroma, to so povečan krvni tlak, diabetes,... Dokazali so namreč. de ima CRP vpliv na povišan krvni tlak, saj so to bolezen odkrili tudi pri ljudjeh, ki so imeli povečan CRP in niso imeli povečan LDL-holesterol, ki je glavni povzročitekj te bolezni. V članku so znanstveniki preučevali zvišane koncentracije človeškega C-reaktivnega proteina v transgenih miših, ki so imele ižražen človeški CRP. raziskovali so kako povečan protein CRP vpliva na razvoj simptomov metaboličnega sindroma. pri miših, kiso imele povečan CRP so ugotovili povečan krvni tlak, vendar je pri vplivu CRP na cel organizem še veliko nejasnosti. Zato bo potrebno še veliko raziskav, da bo znano ali ina človeški C-reaktivni protein neposredno vlogo, pri patogenezi simptomov metaboličnega sindroma

== Urška Navodnik: Stabilnnost DNA/DNA in RNA/DNA dupleksa vpliva na mRNA transkripcijo ==
Nukleinske kisline nam s svojo specifično kemijsko in fizikalno zgradbo zagotavljajo shranjevanje genetskih informacij. Zaradi teh lastnosti imata vijačnici DNA in RNA sposobnost tvoriti medmolekulske interakcije in vodikove vezi, kateri sta glavni razlog, da lahko tvorita dvojno vijačnico – dupleks. Pojavi se vprašanje, če lahko nukleinske kisline posedujejo še druge lastnosti, ki prispevajo k biološkim funkcijam. Eno izmed zanimivih vprašanj se pojavi, ko poskušamo razložiti pojav intron – ekson. Nekateri izmed intronov naj bi imeli vlogo uravnavanja izražanja genov, pa vendar se pri zorenju mRNA izrežejo iz zapisa. V tej raziskavi je predstavljeno, kako lahko termodinamična stabilnost DNA/DNA in RNA/DNA dupleksa vpliva na prepis mRNA. Raziskave so izvajali predvsem na vrsti Saccharomyces z metodo najbližjega soseda. Rezultati so pokazali, da so kodirane regije termodinamsko bolj stabilne od nekodiranih zaporedij – intronov, 3´ - neprevedenih regij in medgenskih področij. Povrh, odprti bralni predelni imajo bolj stabilno smerno RNA/DNA dvojno vijačnico, kot potencialno ujemajoč protismeren dupleks. Raziskava temelji na izračunih, koliko proste energije je potrebno, da se dvojna vijačnica razvije. Več energije, kot je potrebno stabilnejša je struktura. Rezultati torej prikazujejo, da so geni stabilnejši od medgenskih področij. Torej lahko povzamemo, da stabilnost DNA/DNA in RNA/DNA dupleksa vpliva na mRNA prepis.

== Dominik Kert: Kako osteokalcin vpliva na reprodukcijo organizmov ==
Nove raziskave kažejo povezave med reprodukcijo in skeletnim sistemom. Kosti so sestavljene iz osteoblastov. Produkt le teh je hormon osteokalcin. Ta hormon pa zelo vpliva na produkcijo testosterona, ampak ne neposredno. Deluje namreč na leydigove celice, ki se nahajajo med tubuli v testisih. Raziskovalci so odkrivali te rezultate tako, da bo samcem miši vbrizgali osteokalcin-takrat se je raven testosterona dvignila. Po drugi strani pa so miškam odstranili gen, ki kodira ta hormon. Prišlo je do osupljivih rezultatov. Mišim se je zmanjšala plodnost, in sicer tako, da se je zmanjšala količina sperme, zmanjšala število zdravih spermijev in veliko jih je tudi pomrlo že v modih. Dokazali so tudi veliko signalnih poti med reproduciranjem in okostjem. In sicer če organizem dobro funkcionira in ima dovolj zalog hrane se je tudi sposoben razmnoževati. In pa tudi povezavo med debelostjo in neplodnostjo. Ampak to še ni vse. Mišim se je tudi spremenilo partnersko vedenje: manjkrat so postavljali gnezda. To je bila posledica nabiranja luteinizirajočega hormona. Poleg slednjega hormona se je začela pojavljati tudi večja količina estrogena. Te dve posledici pa lahko povežemo s staranjem moškega, ko testosteron začne vpadati, estrogen in luteinizirajoči hormon pa narasteta. Zaradi teh ugotovitev bi bilo lahko v prihodnje možno zdravljenje neplodnosti pri ljudeh.

== Živa Brglez: Kompleks Mre11 ==
Med podvojevanjem dvojne vijačnice DNA včasih pride do napak, ki jih je nujno potrebno popraviti, oziroma obnoviti v njeno izvirno obliko, če je prišlo do poškodbe zaradi mutagenih dejavnikov iz okolja, da se ne prenesejo naprej v hčerinske celice in prihodnje rodove. Za to skrbijo raznovrstni popravljalni mehanizmi. V primeru dvojnega preloma verige DNA (Double-Strand Breaks – DBS) je kompleks Mre11, sestavljen iz treh različnih proteinov: dimera mejotske rekombinacije 11 (Mre11), dimera Rad50 in Nbs1, ključnega pomena za celični odziv na poškodbe DNA. Mre11 in Nbs1 skupaj z delom Rad50 tvorita glavno domeno kompleksa, iz katere izraščajo obvite vijačnice (coiled-coil) Rad50. Poznana sta dva načina poprave preloma dvojne verige DNA, homologna rekombinacija (HR) in sklepanje nehomolognih koncev verig DNA (NHEJ). Kompleks Mre11 sodeluje pri obeh, strukturno in encimatsko. Strukturno tako, da glavno domeno veže poškodovan del DNA, medtem ko se obviti vijačnici povežeta z obvitima vijačnicama drugega kompleksa Mre11 in držita skupaj prelomljene verige. Encimatsko pa s spodbujanjem odstranitve koncev in posredovanjem informacije ATM (ataxia-telengastia-mutated), katerega del se fosforilira na glavni domeni kompleksa Mre11 in tako vpokliče še ostale molekule odgovorne za popravo. Kompleks Mre11 skrbi tudi za homeostazo telomerov z regulira njihove dolžine, in posredno za razvoj imunskega sistema. V primeru mutacije v katerem izmed genov, ki kodirajo gradbene proteine kompleksa Mre11, pride do različnih dednih bolezni, ki se kažejo fenotipsko podobno – značilni sta hiperobčutljivost na radiacijo in mikrocefalija.

== Taja Karner: Alkohol omogoča lažje nalaganje CD1d molekul, s tem aktivira NKT celice in zmanjša pojavljanje sladkorne bolezni pri NOD miškah ==
V raziskavi, ki sem si jo izbrala raziskujejo pozitivne učinke alkohola. Vse več raziskav kaže, da zmerno uživanje alkoholnih pijač pripomore k boljšemu delovanju našega mehanizma. V tej raziskavi pa jih je zanimalo predvsem kakšen je vpliv alkohola na prirojen imunski sistem. Raziskave so potekale in vitro z uporabo α-GalCer molekul in in vivo na NOD miših. NKT celice, ki jih omenjam v svoji seminarski, pa so heterogene skupine, sestavljene iz NK celic in T celic. Te celice prepoznavajo antigene CD1d, ki se izražajo na površini antigen-predstavitvenih celic. To so celice, ki imajo sposobnost preoblikovanja antigenov, tako da jih T-celice prepoznajo in ustvarijo ustrezen odgovor. Ugotovili so, da alkohol izboljša nalaganje CD1d molekul, s tem aktivacijo NKT celic in tako zmanjša možnost za razvoj diabetesa. Pri NOD miših, ki so jim dajali 5 % alkohola se je pokazalo zmanjšano število diabetesa in manjša koncentracija glukoze ob testiranju. Zanimivo je, da te miši niso kazale nikakršnih znakov alkoholizma. Pri ljudeh bi takšna koncentracija, preračunana glede na maso človeka, povzročila visoko stopnjo opojnost. Kljub temu pa je bila v mišji krvi razmeroma mala količina alkohola, kar kaže na bistveno večjo zmožnost razstrupljanja alkohola pri NOD miših, kot jo imamo ljudje.

== Karmen Gec: Učinki vadbe in/ali dodajanja antioksidantov na gene endotelnih celic ==
Teoretično je raziskano, da so antioksidanti v sadju in zelenjavi pomembni pri zaviraju oksidativnih mehanizmov, ki vodijo do različnih degenerativnih bolezni, tudi srčno-žilnih bolezni. V obravnavani raziskavi avtorji na podganah ugotavljajo različno izražanje genov endotelnih celic glede na dodatek antioksidantov, glede na vadbo ali kombinacijo obojega. Navajajo, da je pri ednoteliju zelo pomembno, da razlikujemo med fiziološkimi in patološkimi t.i. Reaktivnimi kisikovimi zvrstmi (v nadaljevanju RKZ). Izražanje genov endotelnih celic so ugotavljali v področju srčnega endotelija (levi ventrikel) in žilnega endotelija (koronarna arterija). V raziskavi so ugotovili, da je gen RhoA, ki je pomemben pri srčno-žilnih boleznih kazal znižan učinek pri vadbi ter povišan pri dodatku antioksidantov v področju levega ventrikla. Poleg tega pa je še IL-6, pomemben gen pri vnetju, znižal učinek pri vseh treh dodanih tretmajih. Tako izražanje obeh genov z dodajanjem vadbe in/ali antioksidantov poda vpogled v molekulske mehanizme srčnožilne bolezni.

== Tjaša Goričan: Molekulske tarče oksidativnega stresa ==
Aerobni organizmi so življensko odvisni od procesov celičnega dihanja, pri čemer pa vedno nastajajo za biološke makromolekule (lipide, DNA, proteine) škodljivi kisikovi radikali. Organizmi so razvili obrambne mehanizme, ki preprečujejo potencialno škodo. Pri tem se ohranja neko ravnovesje med kisikovimi radikali in antioksidanti, katere mora organizem pridobiti s hrano. Antioksidanti (vitamini C, E, koencim Q10, karotenoidi itd.) so snovi, ki delujejo kot katalizatorji in celice varujejo pred oksidacijo. Porušeno ravnotežje povzroči oksidativni stres, posledice katerega so lahko različne bolezni (Alzheimerjeva bolezen, Parkinsonova bolezen, rak, itd.)in staranje. Zmerna oksidacija sproži apoptozo (programirano celično smrt), hujši in intenzivni oksidativni stres pa lahko povzroči celično smrt in celo nekrozo (odmrtje celic/ tkiva). V mojem članku so poskuse izvajali na bakterijah in kvasovkah ter ugotovili, da so pri obeh prvotne tarče ROS (reaktivne spojine, ki vsebujejo atom kisika) različne. Rezultati: pri prokariontih je DNA prvotna tarča, pri evkariontih pa ne. Vzrok za to je različen prag občutljivosti molekul pri različnih organizmih. Identifikacija primarnih tarč oksidativnega stresa bi odprla nove možnosti za terapije bolezni povezane z njim.

== Jernej Mustar: Odpornost srpastih celic na okužbo z plazmodijem ==
V mojem članku so raziskovali fenomen, ki se pojavlja pri obolelih za anemijo srpastih celic(HbS homozigotni) in HbS heterozigotnih, in sicer toleranca do okužbe z Plasmodijem. V raziskavi so uporabili Plasmodium berghei, ki je modelni organizem za razumevanje človeške malarije. Ta povzroča t.i. možgansko malarijo (experimental cerebral malaria-ECM). Znanstveniki so prišli do zelo zanimivih odkritij. Ugotovili so namreč, da se glavni vzrok imunosti skriva v nalaganju nizkih količin prostega hema v krvi in povečane ekspresije stresno-odgovornega encima HemOxigenaze1(ki razgrajuje prosti hem). Pri katalizi hema nastaja CO, ki se veže na hemoglobin in prepreči odcepitev prostetične skupine (hema), saj je le ta glavni vzrok patogeneze ECM. Ta spoznanja so, po mojem mnenju, človeštvo privedla korak bližje k odkritju zdravila za malarijo.
== Ula Štok: Mutacija mitohondrijske DNA v povezavi z rakom debelega črevesa kot posledica abnormalnega delovanja citokroma c oksidaze ==
Mutaciji Ala501Pro in Gly171Asp pri bakteriji ''Rhodobacter sphaerodies'', ki sovpadata z mutacijama Gly125Asp ter Ser458Pro v človeškem telesu povzročata nastajanje rakavih celic, ki vodi do razvoja raka debelega črevesa. Gre za mutaciji v podenoti I kompleksa IV dihalne verige. Procesi dihalne verige so izrednega pomena za proizvodnjo energije, ki jo celica potrebuje za nemoteno delovanje. Elektroni, ki so višek procesa glikolize, se namreč shranjujejo na t.i kofaktorjih in porabljajo kot gonilna sila dihalne verige v mitohondriju. Kompleks IV je predzadnji v tej verigi in je generalno gledano sestavljen iz 13 podenot. Zgradba in sestava vseh podenot še vedno ni povsem jasna/raziskana (predvsem tistih manjših, do 90 aminokislinskih ostankov). Mutacija na podenoti I totalno onemogoči delovanje celotnega sistema. Gre namreč za oviran in predvsem upočasnjen prenos elektronov iz posameznih regij, kar ima za posledico tudi manjšo aktivnost prenosa protonov v intermembranski prostor mitohondrija. Na bolezenski ravni to povzroči nastajanje rakastih celic iz dveh razlogov. Pride do nastanka ROS (Reactive oxygen species), ki so za celico strupeni (v prekomernih količinah) ter do porušenega Δψ / ΔpH razmerja. Sprememba Δψ / ΔpH razmerja povzroči, da se zmanjša transport Ca^2+ ionov v mitohondrij, kar ovira delovanje le-tega, saj so Ca^2+ ioni namreč odgovorni za regulacijo procesov dihalne verige.

BIO1 Povzetki seminarjev 2011

2011-05-29T08:08:43Z

MatjaZalar: /* Ula Štok: Mutacija mitohondrijske DNA v povezavi z rakom debelega črevesa kot posledica abnormalnega delovanja citokroma c oksidaze */

== Alja Zottel: Vloga imunskega sistema pri nastanku ateroskleroze ==
Glavni vzrok nastanka ateroskleroze je imunski odgovor na lipoproteine majhne gostote oz LDL, ki se kopiči pod endotelom arterijskih žil. Apolipoprotein B100, ki je komponenta LDL, se veže na proteoglikane zunajceličnega matriksa in se pod vplivom različnih radikalov oksidira. OxLDL nato aktivira endotelijske celice, da začnejo proizvajati adhezijske beljakovine, kot sta E-selektin in VCAM-1. Te beljakovine skupaj s kemokini povlečejo monocite, T limfocite in in dendritske celice v endotelijsko plast žile. Monociti se nato pod vplivom M-CSF citokina diferencirajo v makrofage. Makrofagi nato začnejo proizvajati odstranjevalne receptorje. Ti tako lahko prepoznajo oxLDL in ga z endocitozo vsrkajo. Makrofagi se zato napihnejo in spremenijo v »foam cell«. Te celice so najštevilčnejše celice v aterosklerotskih plakih. Dejavniki, ki pospešujejo nastanej ateroskleroze so signalni proteini PRR, T levkociti in proteini CRP. T celice pomagalke izločajo interferon gama, ki privlači monocite. Protein CRP se veže na navadni LDL in tako ga lahko makrofagi, ki imajo receptorje za CRP vsrkajo. Dejavniki, ki preprečujejo nastanek ateroskleroze so B limfociti in protein PPAR. PPAR je receptorski protein oz. transkripcijski faktor, ki preprečuje nastanek »foam cell« celic in vsrkavanje LDL v makrofage. Preprečuje tudi razvoj T celic in povečuje količino HDL v krvi.

== Veronika Jarc: Hepatitis C ==
Hepatitis C(HCV) je nalezljiva bolezen, ki napade ljudi, šimpanze ter nekatere majhne modelne živali. HCV spada med RNA viruse z ovojnico.Razvrščen pa je v rod hepacivirus ter družino flaviviridae. Sestavljen je iz 6 genotipov (1-6), ki se razlikujejo v nukleotidni sekvenci od 30-35%, sedmega pa so odkrili leta 2008 (Gottwein et al., 2008). HCV vsebuje pozitiven trak gena (9,6 kb), ki je sestavljen iz 5´-NCR( non-coding region), 3´- NCR in IRES( internal ribosome entry side). IRES vsebuje odprto bralno ogrodje, ki šifrira strukturne in ne strukturne proteine. Med strukturne proteine spadajo proteinsko jedro, virusna RNA ter dva glikoproteina E1 in E2. Sestavni deli ne strukturnih proteinov pa so hidrofoben protein p7, NS2-3 proteaza, NS3 serin proteaza, NS4A polipeptid, NS4B protein, NS5A protein in NS5B RNA odvisna RNA polimeraza (RdRp).
S pomočjo različnih odkritij, kot so HCVpp(sestavljen iz lipidne ovojnice z E1-E2 proteini, na retrovirusni nukleokapsidi), izoliranje kloniranega gena 2a ter s pomočjo tega gena HCVcc( cell-culture produced HCV), so znanstveniki začeli preučevati življenski cikel in celično strukturo hepatitisa C. To so dosegli z preučevanjem različnih eksperimentalnih modelov kot so imunski odzivi, NK celice in dendritske celice.
Poznamo tudi proteine, ki jih HCv sreča v hepatocitski celici in ti so in tegrin RGE/RGD, LDL receptor, HDL receptor, klaudin okludin in tetraspanin CD81.

== Matja Zalar: Protein p53 ==
Protein p53, včasih imenovan tudi varuh genoma, kodira gen TP53 na sedemnajstem kromosomu. Je eden izmed tako imenovanih tumor-supresorskih proteinov, ki, kot to sporoča že samo ime, zavirajo nastanek in rast tumorjev. Na področju razumevanja delovanja, vloge in strukture proteina p53 in njegovih mutantov se izvaja veliko raziskav. Trenutno je p53 najbolj raziskan tumor-supresorski protein, še zdaleč pa ni edini. Gre za protein, ki se kopiči v jedru in z vezavo na DNA v obliki teramera nadzoruje in regulira procese kot so apoptoza, zaustavitev celičnega cikla in popravljanje poškodovane DNA. Za raziskovalce je še posebno zanimiv zaradi dejstva, da v nemutirani obliki zavira nastanek in rast tumojev, njegove GOF mutirane oblike pa pripomorejo k nenadzorovani delitvi celic in nastanku rakastih tkiv. Veliko raziskav se ukvarjaja z iskanjem snovi, ki bi obnovile osnovno obliko p53, oziroma uničile mutantske oblike p53 v rakastih celicah ter s tem uničile tumor. To pa bi lahko bistveno izboljšalo tehnike zdravljenja rakavih obolenj in odziv človeškega organizma na ta zdravljenja. Odkrili so že kar nekaj takšnih snovi (RITA, PRIMA, nutlin3), ki pa jih še vedno testirajo in še niso v redni uporabi pri zdravljenju rakavih obolenj.

== Andrej Vrankar: Androgena alopecija ==
Na podlagi raziskav, ki so jih znanstveniki izvedli na celičnih vzorcih posameznikov z androgeno alopecijo, so ugotovili, da je bila domneva, da je za nastanek AGA kriv propad matičnih celic v lasnem mešičku oziroma, propad samega lasnega mešička napačna. Raziskave so pokazale ravno nasprotno in sicer, da se matične celice tudi v plešastem lasišču posameznika z AGA ohranjajo in da lasni mešički ne propadejo, vendar se le zelo skrčijo. So pa ugotovili, da se število celic imenovanih predniške celice v plešastem lasišču močno zmanjša, kar je eden od glavnih vzrokov za nastanek AGA, saj so prav predniške celice tiste, ki so zaslužene za rast las. Čeprav se dednost smatra kot glavni vzrok za nastanek AGA, pa tudi hormoni igrajo pomembno vlogo. Pri moških je to moški hormon testosteron, ki se s pomočjo encima 5-α-reduktaze v lasno mešičnih celicah pretvarja v svojo bolj aktivno obliko dihidrotestosteron (DHT). Ta se se nato s posebno vezjo veže na androgene receptorje v lasnih mešičkih, kar sproži posebne procese, ki skrajšajo anageno fazo celičnega cikla. Zaradi skrajšanja te faze las prej prestopi v telogeno fazo in izpade. Kako občutljivi so lasni mešički na androgene pa je seveda gensko pogojeno.

== Sandi Botonjić: Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom ==
Kisikovi radikali, ki povzročajo oksidativni stres lahko v skrajnem primeru poškodujejo DNA in tako povzročijo nenadzorovano delitev celic, kar pomeni nastanek raka v organizmu. Hkrati pa je raven kisikovih radikalov v rakastih celicah višja, kot v zdravih, in sicer zaradi onkogenih stimulacij, povečane presnovne aktivnosti ter okvare mitohondrijev. Toda rakave celice imajo, kot protiutež tudi močan antioksidantni mehanizem s katerim zavirajo programirano celično smrt.

Raziskovalci so tekom analiziranja večih tkiv, ki so obolela z različnimi vrstami raka ugotovili, da je pri vseh povečana raven [http://www.thesgc.org/structures/structure_images/2WZ9_400x400.png tioredoksinu podobnega proteina - TXNL2]. Zatem so izvajali poskuse na miših tako, da so jim vbrizgali kancerogene eritrocite in ko so se pojavili simptomi tumorja – so jim vbrizgali še protein TXNL2. Ugotovili so, da protein TXNL2 zavira rast rakavih celic. Proučevali so tudi vpliv proteina TXNL2 v mišjih zarodkih. Prišli so do zaključka, da protein TXNL2 regulira raven kisikovih radikalov tako pri živečih organizmih, kot med embriogenezo. Znanstveniki so prepričani, da je protein TXNL2 potencialna tarča bioloških zdravil v prihodnosti.

== Ana Dolinar: Prilagojena ali prilagodljiva imunost? Primer naravnih celic ubijalk ==
Naravne celice ubijalke (NK celice) so vrsta levkocitov. V človeškem telesu so zadolžene za uničevanje patogenih organizmov s pomočjo za celice strupenih snovi. Na površini imajo pet skupin receptorjev: aktivacijske, inhibitorne, kemotaksične in citokine ter adhezijske receptorje.

Njihova aktivacija je odvisna od vezave ligandov na površinske receptorje NK celice. Če je vezanih več inhibitornih ligandov kot aktivacijskih, potem se NK celica ne aktivira, ker inhibitorni ligandi zavrejo delovanje NK celice. V primeru, da se veže več aktivacijskih kot inhibitornih ligandov ali pa se slednji sploh ne vežejo, se NK celica aktivira ([http://www.georg-speyer-haus.de/agkoch/research/subframe_en.htm aktivirana NK celica-rumeno, tarčna celica-rdeče]). Vezava kemotaksičnih ligandov vpliva na gibanje molekule zaradi kemičnih signalov, vezava citokinov spodbuja rast celic ali sintezo snovi, ki jih potrebuje imunski sistem, vezava adhezijskih ligandov pa omogoča pritrjanje NK celice na tarčno celico.

Raziskovalci se trudijo, da bi našli optimalno imunoterapijo, pri kateri bi sodelovale NK celice. Te terapije bi bile uporabne predvsem pri rakavih obolenjih, vendar so možnosti tudi pri obolenjih z virusom HIV ali z virusom hepatitisa C. Ta način imunoterapije je mogoč, ker večina tumorskih celic in virusov ne izraža MHC tipa 1, pomembnega inhibitorskega liganda za NK celice. [http://media.wiley.com/CurrentProtocols/IM/ima01n/ima01n-fig-0004-1-full.gif Zgradba MHC-1 molekule, prikazana z Ribbonovim diagramom in vezanim peptidom (A) ter površinska struktura molekule z vezanim peptidom (C). Slika B prikazuje molekulo MHC-2 z vezanim peptidom.]

== Urška Rauter: Razvojne vloge Srf, kortikalnega citoskeleta in celične oblike pri orientaciji epidermalnega vretena ==
Mehanizem nastajanja polariziranega epidermalnega sloja, ki s procesoma stratifikacije in diferenciacije tvori kožo, regulira več različnih med seboj v komplekse povezanih bioloških molekul. Trije najbolj osnovni procesi so delovanje proteinov aktina, orientacija vretena in sistem celične signalizacije. Znanstveniki pa so v obširni raziskavi potrdili tudi pomembno vlogo t. i. Srf proteina (serum response factor protein), transkripcijskega dejavnika, katerega pomembna vloga je regulacija celične diferenciacije.

Srf je transkripcijski dejavnik, ki se veže na določen, njemu ustrezen receptorski element; Sre (serum response element), to so predvsem geni v zgodnjem razvoju, geni za razvoj nevronov in mišična gena (proteina) aktin in miozin. Ker je njegova primarna funkcija regulacija ekspresije naštetih genov, odločilno vpliva na celično rast in diferenciacijo, prenos med nevroni in razvoj mišic.

Namen raziskave je obširen. Rezultati obetajoči. Dokazali so pomembno vlogo Srf proteina pri marsikaterem mehanizmu/procesu v embrionalnem razvoju. Tako recimo Srf odločilno vpliva na diferenciacijo celic, saj izguba le-tega povzroči kaotično deljenje in diferenciacijo celic med več plastmi epidermisa. Nadalje vpliva tudi na pravilno vzpostavitev polarnosti bazalne lamine in še najbolj ključno na tvorbo aktinsko-miozinskega skeleta, ki je nujen za pravilno mitozo, posledično za obliko in trdnost celice. Orientacija vretena in asimetrično dedovanje sta po zadnjih raziskavah osrednja mehanizma, ki omogočata matičnim celicam samostojno obnovi in diferenciacijo v pravilni smeri. Rezultati kažejo, da lahko takšne signale pošiljamo preko Srf proteina in aktinsko-miozinskega skeleta, za pravilno tvorbo in nadzirano regulacijo orientacije vretena, asimetrične celične delitve in nasploh usodo posamezne celice. Rezultati razkrivajo nove pojasnitve bioloških procesov, ki sodelujejo pri tvorbi morfologije epidermisa.

== Špela Pohleven: Prioni ==

Prioni so patogeni proteini, ki se od svojih nepatogenih, normalnih, v zaporedju aminokislin enakih dvojnikov, razlikujejo v 3D strukturi – imajo večji del β ploskev. Poznamo več vrst prionov, toda običajno govorimo le o proteinu PrP, ki je prisoten pri ljudeh in živalih. Ostali so namreč značilni za glive, ki so tako primerne za razne raziskave.
Za prione je značilno povezovanje v nitaste polimere, ki jih imenjujemo amiloidi. Znanstveniki domnevajo, da je prav njihova urejena struktura tista, zaradi katere so slabo topni v detergentih in odporni na proteaze.
Najbolj nenavadna lastnost prionov pa je njihova zmožnost širjenja brez potrebe po DNA in RNA. V zvezi s tem potekajo številne raziskave, saj prioni povzročajo številne smrtne bolezni, kot so Creutzfeldt-Jakobova bolezen, smrtonosna družinska nespečnost in druge. Z informacijami, ki jih tako pridobivajo, je možnost za odkritje zdravila večja.
Pri eni od nedavnih raziskav so tako ugotovili, da obstajata dve prionski obliki proteina PrP – infektivna in toksična. Za raziskave so uporabili miši z različnim izražanjem gena PRNP za PrP protein. Vse so okužili s prioni praskavca (ena od prionskih bolezni). Vse so dosegle enak prag infektivnosti, toda smrt ni nastopila istočasno. Iz meritev so znanstveniki prišli do zaključka, da morata obstajati dve različni obliki. To pa je le izhodišče za nove raziskave.

== Maša Mohar: Sladkorna bolezen, kot bolezen imunskega sistema ==

Diabetes mellitus je kronična motnja metabolizma beljakovin, lipidov in ogljikovih hidratov. Nastane zaradi zmanjšane funkcije proizvajanja insulina v telesu. Njen vzrok pa je lahko studi zmanjšana sposobnost telesnih celic za pravilno izkoriščanje insulina. Tip 2 je od insulina neodvisen diabetes (NIDDM). Ta tip ima 80-90% vseh pacientov in se pojavi v odraslem obdobju življenja, spodbudijo ga lahko različni mehanizmi, in za nekatere se še ne ve točno kako pride do tega, je pa res da k temu veliko pripomore nezdrav način življenja in seveda dednost. Prav tako se diabetes tipa 2 deli v dve skupini in sicer na debeli tip, ki ga ima približno 80% vse populacije in na ne debeli tip.
Da je T2D bolezen imunskega sistema pa ugotovimo s tem ko vidimo kako se telo odzovena določene mehanizme, ki sprožijo to bolezen. To so oksidativni stres, stres ER( endoplazemski retikel), lipotoksičnost in glukotoksičnost. Prav tako je potrebno poudariti, da ima diabetes tipa 2 svoje metabolne karakteristike in skupaj s temi patogenimi mehanizmi tvori formulo za nastanek bolezni. Seveda lahko pri T2D pride tudi do dolgoročnih komplikacij, kot so makro in mikro- vaskularne bolezni, problemi z ledvicami, očmi in živci. Te pa so glavni dejavniki za povzročitev hujšega bolezenskega stanja in ne nazadnje tudi smrti zaradi diabetesa.

== Mirjam Kmetič: Regulacija celičnega metabolizma železa ==

Železo je pomemben mikroelement, ki ga vezanega na proteine, vsebujejo skoraj vsa živa bitja. Celice sesalcev potrebujejo zadostno količino železa, da zadovoljijo metabolne potrebe ali dosežejo specializirane funkcije. Vsekakor pa je železo potencialno strupeno, še posebej v obliki Fe2+ ionov, ki katalizirajo pretvorbo vodikovega peroksida v proste radikale, ti pa poškodujejo veliko celičnih struktur (DNA, proteine, lipide...) in posledično celica lahko celo odmre. Vse oblike življenja se temu izognejo tako, da vežejo železove ione na proteine in tako hkrati izkoristijo njegove ugodnosti. Železo se prenaša v tkivo ob pomoči kroženja transferina, prenašalca, ki veže železo v plazmi, katerega predvsem sproščajo črevesne resice in retikuloendotelni makrofagi. Z železom bogat transferin se veže na membranski transferin receptor 1, kar se odraža z endocitozo in sprejemom te kovine. Sprejeto železo se prenese do mitohondrija za sintezo hema ali železo-žveplovih proteinov, ki so bistveni deli mnogih metaloproteinov. Presežno železo se skladišči in detoksificira v feritinu, ki je v citosolu. Metabolizem železa je nadzorovan na različnih nivojih in z raznovrstnimi mehanizmi. Pri uravnavanju je zelo pomemben sistem IRE (iron-responsive element)/IRP (iron-regulatory protein), dobro poznano post-transkripcijsko regulatorno vezje, ki ne le vzdržuje homeostazo v različnih tipih celic, ampak tudi prispeva k sistemskemu ravnovesju železa.

== Lea Kepic: Agonisti adrenoreceptorjev β2 ==

Vloga receptorjev v organizmih je zelo pomembna saj prenaša vse potrebne informacije za delovanje. Delimo jih na ionotropne in metabotropne. Največja skupina metabotropnih receptorjev pripada receptorjem, ki so sklopljeni s proteinom G. Mednje spadajo tudi adrenergični receptorji ali adrenoreceptorji. Adrenoreceptorji so tarčni za katekolamine (fight or flight hormoni) med katere spadajo adrenalin, noradrenalin in dopamin. V svojem seminarju sem se posvetila predvsem podskupini β2 (β2-AR) in njihovim agonistom. Agonisti so spojine, ki se selektivno vežejo na specifične receptorje, ki sprožijo nadaljnji odziv. Njegova naloga je posnemanje naravno obstoječih (endogenih) molekul, kot so na primer hormoni. Najbolj pogost in učinkovit agonist za β2-AR je izoprenalin, med hormoni pa je najboljši adrenalin. S pomočjo eksperimentov znanstveniki raziskujejo posebnosti v zgradbi predvsem kristalnih struktur, tvorbo vezi z različnimi spojinami, konformacijske spremembe, vpliv inhibitorjev, ravnotežna stanja ter energijska pretvarjanja. Rezultati teh raziskav so izhodišče za praktično uporabnost agonistov. Zaradi njihovih lastnosti jih vedno več uporabljamo v medicini za zdravnjenje plujčnih bolezni; predvsem astme in bronhitisa. To področje za enkrat še ni do dobra raziskano zato jih navadno uporabljamo le kot dodatke drugim zdravilom. Raziskani pa so že tudi nekateri negativni učinki na telo.

== Iza Ogris: Zakaj imajo možgani glikogen? ==

Glikogen se v možganih nahaja v precej manjših koncentracijah kot v jetrih in mišicah.Pojavi se vprašanje o njegovi vlogi v možganih in kje se nahaja. Glikogen vsebujejo astrocite- glia celice, ki obdajajo nevrone in skbijo za koncentracijo ionov v izvenceličnem prostoru ter dovajanje določenih snovi nevronom. Ko se med aktivnostjo nevronov v izvenceličnem prostoru kopičijo kalijevi ioni, jih astrocite začnejo privzemati z K/Na ATPazo. Posledično se v astrocitah zviša nivo AMP, kar stimulira delovanje encima glikogen fosforilaze (razgradnja glikogena). Astrocite med nevronsko aktivnostjo privzemajo tudi živnčni prenašalec glutamat iz sinaps, ki tudi posredno povzroča padec energije v astrocitah. Ko se nivo glukoze v dejavnih nevronih znižuje, se medtem v astrocitih povečuje. Koncentracija glukoze je nato v astrocitih večja kot v izvencelični tekočini in nevronih, zato se ustvari koncentracijski gradient kar omogoči pot glukoze iz astrocitov v nevrone. Pri vzdrževanju glukoze se tako razgradnja glikogena izkaže za bolj učinkovito kot le privzem glukoze iz krvi. Razkriva se izvor in usoda glukozne rezerve.

== Ines Kerin: Kanabinoidi za zdravljenje shizofrenije? Uravnotežena nevrokemična sestava za škodljive in terapevtske učinke uživanja konoplje ==

Že desetletja velja prepričanje, da je uživanje konoplje eden pomembnih dejavnikov za nastanek in razvoj shizofrenije. Vendar so v novejših raziskavah odkrili, da naj bi kanabinoidi, psihoaktivne substance v konoplji, izboljšali nevropsihološke učinke in negativne simptome, ter imeli antipsihotične lastnosti pri ljudeh s shizofrenijo. Shizofrenija je huda duševna bolezen iz skupine psihoz. Simptome shizofrenije povzroča spremenjena količina določenih snovi v možganih, in sicer živčnih prenašalcev, ki omogočajo medsebojno komunikacijo možganskih celic. Motnje v komunikaciji pa povzročajo spremembo v delovanju možganov. Pomembno vlogo ima pri bolezni dopamin, ki lahko s prevelikim sproščanjem izzove nekatere simptome.
Shizofrenijo zdravijo s pomočjo antipsihotikov, ki imajo podobne lastnosti kot kanabinoidi v konoplji. Vendar se učinki konoplje od učinkov antipsihotikov nekoliko razlikujejo. Pri negativnih simptomih konoplja, tako kot antipsihotiki, spodbuja sproščanje in delovanje dopamina. Manj znano pa je, ali zavira ali spodbuja delovanje ostalih petih nevrotransmiterjev (serotonina, acetilholina, noradrenalina, glutamina in GABA). Na pozitivne simptome ima konoplja, kot je vidno v tabeli lahko tako koristne kot nekosristne učinke. Simptome lahko izboljša z zaviranjem sproščanja serotonina, acetilholina in noradrenalina. V primeru dopamina, glutamata in GABA ima konoplja negative učinke, saj v nasprotju z antipsihotiki, poveča sproščanje dopamina in zavira delovanje glutamata in GABA. Obstajajo dokazi, da imajo kanabinoidi zdravilne učinke na pozitivne in negativne simptome pri shizofreniji. Vendar to poglavje še ni zaključeno in se izvajajo še nadalnje raziskave v tej smeri.

== Eva Knapič: Kako virusi vodijo delovanje celice. ==
Virusi so geni obdani z zaščitno proteinsko ovojnico. Za izražanje teh genov, da lahko naredijo proteine in podvojijo kromosome, je potrebno, da vstopijo v celico in uporabijo celične mehanizme, saj sami tega niso zmožni. Poznamo več vrst virusov. Posebnost evkariontskih virusov je sposobnost posnemanja kratkih linearnih motivov proteinov poznanih pod kratico SLiMs. To so deli proteinov, ki so odgovorni za posredovanje med nekaterimi celičnimi funkcijami. So zelo kratki, večinoma nekje od 3 do 10 aminokislin. Motivi sodelujejo pri vezavi proteinih, pri prepoznavanju post-translacijske modifikacije encimov, pri usmerjanju proteinov v celične razdelke in pa so prisotni na cepitvenih mestih proteina. S posnemanjem različnih motivov lahko virusi prevzamejo nadzor nad celico. Najpogostejši mehanizmi prevzema nadzora so uporaba celičnega transporta, manipuliranje signalnega transporta, nadzor proteinov v celici, regulacija prepisovanja, sprememba modifikacije gostiteljevega proteina in usmerjanje modifikacije proteinov.
Uporaba proteinskih motivov v celici in lahko posnemanje le teh predstavlja šibkost v celični organiziranosti, saj virusi s pridom izkoriščajo to v svojo korist. Posnemanje motivov virusom omogoča, da sami vodijo delovanje celice in se sami s pomočjo celičnih mehanizmov enostavno razmnožujejo in tako hitro okužijo celoten organizem.
V nadalje bodo potekale raziskave za izkoriščanje posnemanja motivov v namene zdravljenja virusnih okužb.
== Katra Koman: Pomen dendritskih celic (DCs) in celic ubijalk (NK) v imunskem odzivu na okužbo z virusom HIV-1 ==
Dendritske celice (DCs - dendritic cells) in celice ubijalke (NK – natural killer cells) sta dva tipa celic prirojenega imunskega sistema, ki imata zelo pomembno vlogo pri protivirusni odpornosti. Tako dendritske celice, kot tudi celice ubijalke so sicer pomemben del (nespecifičnega) prirojenega imunskega sistema, a hkrati vplivajo tudi na učinkovit razvoj (specifičnega) prilagojenega imunskega odziva. DC so ključnega pomena za aktiviranje za virus specifičnih T celic, kar pa je močno odvisno od prejšnjega, prirojenega imunskega odziva. NK celice pa ovirajo zgodnje širjenje virusov, tako da proizvajajo citokine in s fagocitozo neposredno uničujejo okužene celice. Razumevanje delovanja in funkcije teh celic pa ima pomemben vpliv na razvijanje nove strategije cepiva proti virusu HIV-1, katere uspeh bo odvisen od primernega razumevanja delovanja teh celic.

== Jana Verbančič: Apoptozi podobna smrt v bakterijah, ki jo povzroča HAMLET, lipidno-proteinski kompleks v človeškem mleku ==
Apoptoza oz. programirana celična smrt je eden najpomembnejših procesov v evkariontskih celicah. Organizem je z apoptozo sposoben sam uravnavati število živih celic. Uniči jih lahko, ker so poškodovane, stare ali ker ne opravljajo več svoje naloge, lahko pa uniči tudi popolnoma zdrave celice, ki jih ne potrebuje več (npr. pri embrionalnem razvoju). Pomemben dejavnik pri apoptozi so encimi kaspaze, ki cepijo in aktivirajo druge proteine, vse skupaj pa lahko poteka po dveh poteh. Prva je notranja in vključuje mitohondrije in citokrom c, ki deluje kot signalna molekula v apoptotskem ciklu ter tako sproži delovanje kaspaz in posledično apoptozo. Druga pot je zunanja in vključuje aktivacijo proteinskih receptorjev (t. i. receptorjev smrti) na zunanji strani membrane. Oblikuje se kompleks iz receptorja, adaptorskega proteina in vezane kaspaze (DISC), ki povzroča cepljenje in aktivacijo nadaljnjih kaspaz; to pa spet vodi v apoptozo. V mehanizme so lahko vključeni mnogi drugi proteini ali neproteinski signali.
Programirane celične smrti pa nimajo samo evkarionti, ampak so dokazali, da so tudi prokarionti sposobni procesov, ki so zelo podobni apoptozi. Raziskave so delali na streptokokih in tumorskih celicah, ki so jim dodali kompleks HAMLET (human alpha-lactalbumin made lethal to tumor cells), ki ga lahko najdemo v človeškem mleku. Kompleks je deloval kot signalna molekula za začetek apoptoze v tumorskih celicah oz. za začetek apoptozi podobnega procesa v bakterijah.

== Ana Remžgar: Črevesna absorpcija vitamina D ne poteka le s pasivno difuzijo: dokazi za vpletenost enakih transporterjev kot pri holesterolu ==
Vitamin D je hormon, ki ga telo lahko proizvede samo s pomočjo obsevanja kože z ultravijolično svetlobo, vendar je hipovitaminoza D razširjena v mnogih državah in je pomemben svetovni zdravstveni problem. Vitamin D je nujno potreben za uravnavanje ravnovesja med kalcijem in fosfati v telesu ter za normalno rast kosti.

Dolgo časa je veljalo, da se v črevesju vitamin D absorbira le s pomočjo pasivne difuzije. Znanstveniki so kulturi človeških embrionalnih ledvičnih (HEK) celic dodali vsaj enega od teh membranskih proteinov (SR-BI, CD36, NPC1L1). Ti trije proteini so pomembni pri absorpciji holesterola. Zaradi podobne zgradbe holesterola in vitamina D, so znanstveniki sklepali, da so lahko ti trije proteini pomembni tudi pri absorpciji vitamina D. Ko so HEK celicam dodali te proteine, se je absorpcija vitamina D opazno povečala. Ko pa so HEK celicam dodali poleg proteinov še njihove inhibitorje, se je absorpcija močno zmanjšala.
Vpliv SR-BI so opazovali tudi in vivo. Uporabili so wild type miši ter miši z mnogo bolj izraženim Scavenger receptorjem razreda B tipa I (SR-BI). Tudi tu se je pri SR-BI miših povečala absorpcija vitamina D.

Ti rezultati nam prvič pokažejo, da se vitamin D v črevesju ne absorbira le preko pasivne difuzije vendar je v ta proces vključenih kar nekaj transporterjev.

== Maja Grdadolnik: Jedrni in nejedrni receptorji za estrogene. ==
Receptorji za estrogene oz. estrogenski receptorji so proteinske molekule z vlogo specifičnega mesta vezave ustreznega liganda. Nahajajo se v vseh celicah tkiv, ki so tarčne celice estrogena. Lahko se nahajajo v jedru celice, v neposredni bližini DNA, lahko pa so vezani na posebna mesta na membrani celice, t.i. caveole.
Estrogeni (estron (E1), estradiol (E2), estriol (E3)) so lipidopolarni in brez večjih težav prehajajo skozi lipidni dvosloj. Nato se vežejo na lipoproteine v krvi, ki jih prenesejo do jedra tarčne celice. Tarčne celice so po navadi celice jajčnikov, testisov, nadledvičnih žlez, jeter in prsi. V jedru se nato vežejo na estrogenski receptor, s katerim tvorijo kompleks. Ta ligand-receptor kompleks se nato s posebnim mestom (domeno E) veže na specifično mesto na DNA, imenovano estrogen response element (ESE). S tem sodeluje pri procesu transkripcije in uravnava sintezo ustreznih proteinov.
Nejedrni estrogenski receptorji so vezani na posebna mesta na membrani celice, t.i. caveole. Na ta mesta so vezani z integralnim proteinom, za vezavo pa potrebujejo aminokislinski substrat. Receptorje na membrani lahko povezujemo z interakcijo z različnimi ligandi, imajo pa tudi pomembno vlogo posredne aktivacije endotelijske NO sintaze, ki pozitivno vpliva na srce in ožilje. Nejedrne estrogenske receptorje že povezujejo s procesi, ki blagodejno vplivajo na kardiovaskularne bolezni in tkivo endotelija.

== Andreja Bratovš: Vloga GPCR v patologiji Alzheimerjeve bolezni. ==
Alzheimerjeva bolezen je najpogostejša oblika demence. Zaradi odmiranja nevronov pride do zmanjšanja obsega možganov in pešanja razumskih funkcij. Eden glavnih razlogov za nastanek bolezni so amiloidni plaki. Ti nastajajo s kopičenjem amiloidnih peptidov beta. APP (amyloid precursor protein) je membranski protein, ki ga pri zdravem človeku cepi najprej α-sekretaza (nastane sAPPα), nato pa še γ-sekretaza – nastane topen delec p3. Kadar pa APP cepi β-sekretaza, nastane najprej sAPPβ, po cepitvi z γ-sekretazo pa nastane amiloidni peptid beta.
Pri iskanju zdravila za Alzheimerjevo bolezen se trenutno osredotočajo prav na amiloidne plake oz. na preprečevanje njihovega nastajanja ter njihovo razgradnjo. Alternativen pristop imunoterapiji je regulacija receptorjev, sklopljenih z G-proteini, saj so ti udeleženi v več fazah nastajanja plakov. Možnih je več poti, in sicer: zaviranje nastajanja amiloidnih peptidov beta z regulacijo α-, β- ali γ-sekretaze ter sproščanje encimov za razgradnjo plakov. Pri regulaciji α-sekretaze gre za promoviranje njenega delovanja, saj se tako poleg tega, da ne nastajajo amiloidni peptidi beta, tudi sprošča sAPPα, ki ima vlogo pri zaščiti nevronov. Za β-sekretazo je sicer znanih veliko inhibitorjev, vendar jih iz možganov eksportira P-glikoprotein. Problem pri γ-sekretaze je, da ta sekretaza cepi tudi del proteina Notch, zato bi z njeno inhibicijo vplivali tudi na Notch signalno pot.

== Kaja Javoršek: Potencial matičnih celic pri Parkinsonovi bolezni in molekularni faktorji za tvorbo dopaminskih nevronov. ==
Parkinsonova bolezen je nevrodegenerativna bolezen bazalnih ganglijev. ta bolezen prizadane predvsem telesno gibanje, nastane pa ker se zmanjša koncentracija dopamina v striatumu. Kot posledica tega, začnejo propadati dopaminski nevroni v substanti nigri. Prav propadanje dopaminskih nevronov pa je vrzok za Parkinsonovo bolezen. Vzrok za propadanje teh nevronov pa še vedno ni znan. Znano je da dopaminski nevroni s starostjo pospešeno propadajo. To je tudi razlog, zakaj se ta bolezen pojavlja šele pri starejših ljudeh.
Danes se v medicini uporablja veliko terapij, ki pa le lajšajo simptome in bolezni ne pozdravijo. Prav to je razlog za tako veliko število raziskav povezanih s Parkinsonovo boleznijo. Čeprav mehanizmi razvoja dopaminskih nevronov še niso povsem znani, so raziskovalci odkrili kar nekaj molekularnih faktorjev, ki vplivajo na njihovo tvorbo, na primer Fox proteini in receptor sirota Nurr1. Fox proteini so transkripcijski faktorji, ki vežejo DNA. Med temi proteini igrata najpomembnejšo vlogo v nastanku dopaminskih nevronov FoxA1 in FoxA2 proteina. Receptor sirota Nurr1 pa je pomemben pri nastanku L-DOPE, ki je vmesen produkt pri nastanku dopamina iz L-tirozina. Za nastanek L-DOPE mora biti prisoten encim tirozin hidroksilaza. Za izražanje tega encima pa je pomemben receptor sirota Nurr1 in mutacije tega receptorja so povezane s Parkinsonovo boleznijo in shizofrenijo.
Poleg vseh načinov zdravljenja, pa poskušajo Parkinsonovo bolezen pozdraviti tudi s pomočjo matičnih celic, saj so se zmožne diferencirati v katero koli vrsto celic, vključno z dopaminskimi nevroni.

== Tamara Marić: Organizacija jedra. ==
Organizacija genoma v jedru je zelo kompleksna in dinamična in prav to je znanstvenike privedlo do mišljenja, da ima jedro neko globjo strukturo, kjer mora vladati red. S pomočjo novih tehnoloških metod (3C, FISH, 4C) so odkrili kar nekaj zanimivh stvari o sestavi samega jedra. Jedro si moramo predstavljati kot 3D strukturo, v kateri se neprestano nekaj dogaja. Sestavljen je iz dveh glavnih domen, obrobja in centra. Na obrobju sta še dva pododdelka. Ob jedrnih porah se nahajajo aktivni geni, ki so povezani s številnimi proteini, speči geni pa se nahajajo ob lamini. V centru se pododdelki medseboj razlikujejo po funkcijah. V jedrcu se nahajajo geni za rRNA, v transkripcijskih tovarnah se nahajajo vse »sestavine«, ki jih geni potrebujejo za prepis, polycombska telesca imajo bistveno vlogo pri ohranjevanju represije in perinuklearni prostor je specializiran za replikacijo heterokromatina. Ker pa to ne miruje je logično da kromatinske zanke med seboj interagirajo. Poznamo homologne (kjer gre za podobno zgrajene/iste kromosome) in nehomologne(se med seboj razlikujejo) interkromosomske interakcije. Pri prvi je pomembno, da se podobna kromosoma »zmenita«, kateri bo aktiven, pri drugi pa je diferenciacija celice odvisna od aktivnosti nekega gena.

== Mitja Crček: Matične celice in njihova vloga pri zdravljenju bolezni in poškodb. ==
Regeneracija je proces, pri katerem nadomestimo poškodovane telesne dele. V človeškem telesu imajo to nalogo matične celice (MC), ki skribijo za delno regeneracijo in celjenje poškodb. Matične celice so nediferencirane celice odraslege človeka ali zarodka, ki imajo izjemen potencial, da se defirencirajo v mnogo različnih tipov celic v telesu. V tri do pet dni starih zarodkih iz matičnih celic nastane celotno telo organizma, pri odraslih ljudeh pa nas matične celice ohranjajo pri življenju. Glede na potentnost jih razdelimo v štiri razrede: totipotentne in pluripotentne MC so celice, ki se lahko diferencirajo v praktično vse celice telesa, medtem ko so multipotentne in unipotentne bolj omejene. Drugo delitev lahko opravimo glede na njihov izvor: embrionalne MC izvirajo iz zarodka, medtem ko MC odraslih tkiv in organov najdemo med že diferenciranimi celicami. Zaradi vseh njihovih lastnosti imajo velik potencial pri zdravljenju bolezni, že vrsto let jih uporabljajo za zdravljenje levkemije in limfoma. Z diferenciacijo MC v nevrone bi lahko pozdravili poškodbe hrbtenjače in možganov, ob sproščanju kemičnih signalov iz MC proti lasnim mešičkom bi lahko pozdravili plešavost. V teoriji bi lahko nadomestili tudi izgubljen zob, zdravili slabovidnost in gluhost, pa tudi sladkorno bolezen in neplodnost. Velik potencial imajo tudi pri zdravljenju poškodb kosti in mišic. Pri zlomih ter poškodbah hrustanca in vezi služijo predvsem za hitrejšo regeneracijo, omogočajo pa tudi zdravljenje mišične distrofije ali pa povečanje mišične mase in moči, kar bi lahko s pridom izkoriščali športniki in starejši ljudje.

== Sara Lorbek: Sovplivanje maščobnih kislin ter genov na adipokine in debelost. ==
Belo maščobno tkivo ni namenjeno zgolj shranjevanju zalog maščobe, temveč ima velik vpliv na prisotnost in stopnjo vnetja v telesu, saj na le-to vpliva s sekrecijo adipokinov. Adipokini so proteini, ki se izločajo iz celic maščobnega tkiva, do danes pa je poznanih že več kot 100 različnih. Njihov vpliv je zelo različen, v nalogi pa sem se osredotočila na vpliv aipokinov na vnetje, za katerega velja, da ga sproža debelost. Adipokina, ki odločilno prispevata k vnetnemu stanju sta TNF in interlevkin-6 (IL-6), njuna količina pa je močno odvisna tudi od telesne teže: večja kot je telesna teža posameznika, več je teh dveh adipokinov, ki promovirata vnetje, zato je tudi stopnja vnetja večja pri debelejših osebkih. Maščobne kisline veljajo za snovi, ki so sposobne regulirati proizvodnjo adipokinov in s tem vplivati na stopnjo vnetja, toda natančni molekulski mehanizmi tovrstne aktivnosti maščobnih kislin še niso pojasnjeni. Kljub temu imamo že dovolj dokazov, da lahko z gotovostjo trdimo, da različni tipi maščobnih kislin različno ugodno/neugodno vplivajo na promocijo vnetja v organizmu, tako npr. uživanje večkrat nenasičenih in omega-3 m.k. znižuje količino IL-6 in TNF- torej zavira vnetje, uživanje nasičenih m.k. pa vnetje promovira, saj zvišuje količino IL-6 in TNF v organizmu. Odziv številnih adipokinov na različne m.k. do danes še ni bil raziskan, kar predstavlja nov izziv za področje nutrigenomike.

== Maja Remškar: Evolucijska dinamika transponibilnih elementov v majhnem RNA svetu ==
Genom si pogosto predstavljamo kot nekaj statičnega, a ni tako. V zadnjem času so odkrili transponibilne elemente, samostojne dele DNA, ki se lahko premeščajo po genomu in sprožajo mutacije. Če se vgradijo v v stukturne gene, običajno uničijo njihovo informacijo, če pa se vgradijo v regulacijske regije vplivajo na izražanje genov, navadno jih naredijo neaktivne. Vsebujejo gene za podvajanje in premeščanje. Sestojijo iz obrnjenih ponovitev na vsakem koncu in iz vsaj še gena za transpozazo, ki mu omogoča premeščanje. Vmes imajo lahko poljubno število genov. Transpozoni naj bi bili odvečna in sebična DNA in dolgo je bila naravna selekcija edini poznan pojav, ki je nadzoroval njihovo pretirano razmnoževanje. Dandanes vemo, da je mehanizmov njihovega zaviranja več, in sicer, lahko delujejo samorepresorsko (za vrste, ki se razmnožujejo nespolno), pri zaviranju lahko pomaga represorski alel, ki je navadno s škodljivimi TE v stiku, lahko pa jih nadzorujeta mehanizma siRNA in piRNA, ki vztrajno popravljata napake povzročene s strani transpozonov. Na dinamiko represorskih alelov vpliva genetski zdrs – zaradi zmanjšanja populacije, se poruši naravno ravnovesje in lahko pride do fiksacije škodljivejših alelov – in rekombinacija, ki prekine povezave med represorskimi aleli in njihovim tarčnim mestom vezave ter prepreči njihovo fiksacijo. Transpozone so preučevali na koruzi, v bakterijah, vinski mušici in tudi pri človeku, saj povzročajo dedne bolezni kot sta hemofiliji A in B ter Duchennova mišična distrofija.

== Rok Štemberger: virus HIv in povečana ekspresija ter imunogenost HIV-1 proteaze po deaktivacija encimske aktivnosti ==
Virus HIV spada v skupino retrovirusov in je povzročitelj ene najhujših svetovnih pandemij, ki vsako leto terja skoraj 3 milijone žrtev. Virus HIV potrebuje za svoje razmnoževanje gostiteljsko celico, ker nima svojih lastnih mehanizmov, s katerimi bi se lahko razmnoževal. Njegov razmnoževalni cikel obsega veliko procesov, ki se morajo izvršiti, da se virus HIV lahko ustrezno replicira. V moji raziskavi je bil pod drobnogled vzet eden izmed treh encimov, ki sodelujejo pri razmnoževanju HIV-a, in sicer HIV-1 proteaza. HIV-1 proteaza je encim, ki dolge verige proteinov cepi na manjše dele. Če se HIV-1 proteazo z inhibitorji blokira, bi to pomenilo da virus HIV ne bi imel potrebnih encimov za svoje razmnoževanje, saj je dolga veriga proteinov popolnoma neuporabna, če niso razrezani na manjše dele. V raziskavi so ugotovili, da če uporabimo mutirano HIV-1 proteazo, se ji aktivnost drastično zmanjša po drugi strani pa so opazili veliko ekspresijo. Ta ekspresija se je pokazal v tem, da je inducirala imunski odziv in HIV-1 proteaza je bila trača predvsem CD8+ T celic pomagalk. Kasneje so ugotovili, da lahko prav te T celice popolnoma uničijo mutirano HIV-1 proteazo iz telesa in jo s tem odstranijo iz našega sistema. Poskusi so bili narejeni tudi na transgenih miših, ki dajejo bolj verodostojne rezultate kot ostale miši. Ta raziskava bo osnova vsem nadaljnjim raziskavam, ki se bodo ukvarjali predvsem z HIV-1 proteazo, saj ta do sedaj ni bila deležna velike pozornosti. Različni Inhibitorji HIV-1 proteaze pa bodo v prihodnosti bili še bolj pogosto uporabljeni v mešanici zdravil proti bolezni HIV.

== Rok Vene: Spremembe nivoja metilacije DNA so sorazmerne s starostjo človeških možganov ==
V zaporedju DNA se nahajajo posebna zaporedja nukleotidov, ki so edina, na katerih lahko poteče metilacija. Ta mesta sestavlja dinukleotid CpG – cytosine-phosphat-guanine (Od 5' konca DNA verige proti 3' koncu sta citozin in gvanin zaporedno vezana s fosfodiestersko vezjo – 5'-CG-3'). Metilacija DNA je proces v katerem se na ta posebna t.i. CpG mesta veže metilna skupina (-CH3). Taka mesta so v DNA redkejša, kot bi statistično gledano smela biti. Večina CpG mest je metiliranih. CpG mesta se lokacijsko na DNA lahko nahajajo v skupkih imenovanih CpG otočki (CpG islands), ali posamič. Direktna posledica metiliranih CpG mest je utišanje genov, na katerih se ta metilirana mesta nahajajo. Skupaj z ostalimi epigenetskimi faktorji pa indirektno vplivajo še na diferenciacijo celic. Destabilizacija epigenetskih faktorjev je lahko vzrok za številne bolezni (rak, sindromi Rett, ICF, Prader-Willi,...).

V raziskavi iz članka so znanstveniki primerjali količino in lokacijo metilirane DNA v različnih možganskih tkivih. Precejšen del rezultatov je specifičen glede na vrsto tkiva (preko petsto lokusov), vendar obstajajo določene povezave, ki so značilne za vsa raziskovana tkiva možganov. Odkrili so deset specifičnih lokusov, ki vsi vsebujejo metilirana CpG mesta. Odkrili so tudi, da nivo metilirane DNA s starostjo tkiva narašča. Starost tkiva je bila v kar 32-75% primerov glavni razlog za spremembe v količini metilirane DNA. Nekatere izmed destih lokusov so že pred to raziskavo povezovali z starostnimi spremembami v metilaciji DNA, vendar so šest izmed desetih najpomembnejših lokusov odkrili na novo.

== Karmen Hrovat: Ciljanje kemokinih receptorjev v alergijskih boleznih ==
Kemokini so družina majhnih proteinov, velikosti 8-10 kDa. Sodelujejo v procesu agiogeneze, embriogeneze, za nas pa je bistveno, da spodbujajo premikanje levkocitov, bazofilcev, monocitov, jih usmerjajo in nadzorujejo njihov prehod iz krvi v tkiva. Dandanes jih uvrščamo med mnoge raziskave, povezujemo jih tako z aterosklerozo, prenosljivimi boleznimi kot sta virus HIV in malarija, rakom, luskavico in alergijskimi boleznimi med katere sodijo astma, alergijski rinitis in atopijski dermatitis. Poznamo štiri vrste kemokinov: CC,CCX, C in CX3C. Nekateri kemokini se vežejo na več različnih receptorjev in obratno.V članku je opisanih več poskusov na kemokinih receptorjih v miših obolelih za alergijskimi boleznimi.

Farmacevti si prizadevajo odkriti kemokine receptorje antagoniste, saj se je aktivacija GPCR kompleksa izkazala uporabna za zdravilo. V miših obolelih za alergijskimi boleznimi je bilo do sedaj tako in vitro kot tudi in vivo dokazanih že veliko antagonistov kemokinih receptorjev. Kljub temu številni zaradi vprašanja varnosti in farmacevtskih dogm niso dočakali kliničnega sojenja.Lahko rečemo, da napredek ovira tudi pomanjkljivo razumevanje funkcije kemokinov in njihovih receptorjev v alergijskih boleznih. Kljub temu pa se bodo v prihodnost namenili k iskanju antagonistov potencialnega kandidata kemokinega receptorja CCR3.

== Matevž Ambrožič: Termogene snovi in regulacija telesne teže ==
Eden izmed glavnih problemov modernega človeka je prekomerna teža in z njo povezane zdravstvene težave. Strokovnjaki so si edini, da je ključ do uspeha pozitivno razmerje med porabljeno in vnešeno energijo. Veliko pomoč pri porabi energije nudijo snovi, ki spodbujajo termogenezo, po možnosti prek oksidacije maščob. Katehini in kofein so termogene snovi, ki jih najdemo v mnogih naravnih virih, vsi skupaj pa se pojavljajo v čaju. Najboljša vira sta zeleni in beli čaj, saj sta manj obdelana. Pri izgubi telesne teže je vedno cilj večja poraba energije in uničevanje maščobnih zalog. Maščobe, ki jih zaužijemo, so v obliki triacilglicerolov in se shranjujejo v belem in rjavem maščobnem tkivu. Služijo nam kot rezervna zaloga energije, mehanska zaščita in pomoč pri vzdrževanju temperature. V določenih pogojih simpatični živčni sistem z izločanjem hormona epinefrina sproži pretvarjanje shranjenih triacilglicerolov v maščobne kisline (proces lipolize), te pa se lahko v procesu oksidacije porabijo za sintezo ATP. Da pospešimo porabo maščob, se s termogenimi snovmi skušamo vmešavati v metabolizem maščob na raznih stopnjah. Katehini in kofein katalizirajo lipolizo na različne načine, večinoma z inhibicijo zaviralcev lipolize. Rezultati raziskav sicer rahlo variirajo, vendar v splošnem velja, da katehini in kofein pomagajo pri regulaciji telesne teže.

== Marko Radojković: Vpliv rakastih celic in sepse na izraženost krvnega proteina trombina ==
Nedavne študije so pokazale kako anti-koagulanti pomagajo pri zdravljenju in preprečevanju raka, vendar natančen mehanizem ki opisuje kako sta strjevanje krvi in napredovanje raka povezana, ni bil znan do sedaj. Znastveniki so odkrili kako celice pod stresom povečajo proizvodnjo enega izmed ključnih faktorjev strjevanja krvi – trombina. Količina trombina ki ga naše celice proizvajajo je kontrolirana z dvema vrstami proteinov : proteini ki zavirajo produkcijo (FBP2 in FBP3) , ter proteini ki jo pospešujejo (hnRNPI, U2AF65 in U2AF35). Obe vrsti proteinov delujeta tako da se vežeta na celične ''stroje'' na protrombinski mRNA , in v normalnih pogojih, proteini inhibitorji vzdržujejo nizko koncentracijo trombina. Ko naše celice pridejo v stanje stresa, v primeru ko je povzročitelj vnetje, en drugi protein ki se imenuje p38 MAPK, reagira tako da pripne fosfatne skupine ihibitornim proteinom. To povzroči da se le ti težje vežejo na celične ''stroje'' za produkcijo trombina, in omogoča stimulatornim proteinom da prevzamejo glavno vlogo v mehanizmu. Torej, vnetje zaradi raka bi lahko pripeljalo do povečane ravni trombina in, kot je trombin glavni agent strjevanja krvi, bi to lahko pojasnilo, zakaj se pri bolnikih z rakom pogosteje pojavljajo krvni strdki.
Znastveniki so ugotovili da p38 MAPK protein tudi vpliva na proizvodnjo trombina v sepsi. Znana tudi kot zastrupitev krvi, sepsa se pojavi, ko bakterije ali drugi povzročitelji bolezni pridejo v kri, ki vodi k razširjenosti okužbe in nastanku težav pri strjevanu krvi. Ko so analizirali vzorce jeter odvzetih iz miši s sepso in iz bolnikov z rakom, so znanstveniki odkrili da je povečana produkcija trombina odgovor, tako kot na široko vnetje v primeru sepse, kot na lokalizirano vnetje v invaziji tumorja , kje se rakave celice širijo v bližnje tkivo.

== Urška Rode: Vpliv C- reaktivnega proteina na patogenezo simptomov metaboličnega sindroma ==
C-reaktivni protein je protein, akutne faze, ki nastaja v jetrih, njegova koncentracija v krvi se poviša ob razih vnetjih in okužbah, kot del imunskega odziva. Njegova vloga in pomen še nista povsem jasna. Najbolj znana njegova vloga je pri vezavi na fosfoholin na membrano bakterije ali poškovodovane celice. s tem ko se veže z eno strenjo na fosfoholin se na njegovo drugo stran veže prva komponenta klasične proti imunskega odziva.
njegova vloga na cel organizem še ni popolnoma pojasnjena. V zadnjem času poteka veliko raziskav, ki poučujejo njegov vpliv na sindrome metaboličnega sindroma, to so povečan krvni tlak, diabetes,... Dokazali so namreč. de ima CRP vpliv na povišan krvni tlak, saj so to bolezen odkrili tudi pri ljudjeh, ki so imeli povečan CRP in niso imeli povečan LDL-holesterol, ki je glavni povzročitekj te bolezni. V članku so znanstveniki preučevali zvišane koncentracije človeškega C-reaktivnega proteina v transgenih miših, ki so imele ižražen človeški CRP. raziskovali so kako povečan protein CRP vpliva na razvoj simptomov metaboličnega sindroma. pri miših, kiso imele povečan CRP so ugotovili povečan krvni tlak, vendar je pri vplivu CRP na cel organizem še veliko nejasnosti. Zato bo potrebno še veliko raziskav, da bo znano ali ina človeški C-reaktivni protein neposredno vlogo, pri patogenezi simptomov metaboličnega sindroma

== Urška Navodnik: Stabilnnost DNA/DNA in RNA/DNA dupleksa vpliva na mRNA transkripcijo ==
Nukleinske kisline nam s svojo specifično kemijsko in fizikalno zgradbo zagotavljajo shranjevanje genetskih informacij. Zaradi teh lastnosti imata vijačnici DNA in RNA sposobnost tvoriti medmolekulske interakcije in vodikove vezi, kateri sta glavni razlog, da lahko tvorita dvojno vijačnico – dupleks. Pojavi se vprašanje, če lahko nukleinske kisline posedujejo še druge lastnosti, ki prispevajo k biološkim funkcijam. Eno izmed zanimivih vprašanj se pojavi, ko poskušamo razložiti pojav intron – ekson. Nekateri izmed intronov naj bi imeli vlogo uravnavanja izražanja genov, pa vendar se pri zorenju mRNA izrežejo iz zapisa. V tej raziskavi je predstavljeno, kako lahko termodinamična stabilnost DNA/DNA in RNA/DNA dupleksa vpliva na prepis mRNA. Raziskave so izvajali predvsem na vrsti Saccharomyces z metodo najbližjega soseda. Rezultati so pokazali, da so kodirane regije termodinamsko bolj stabilne od nekodiranih zaporedij – intronov, 3´ - neprevedenih regij in medgenskih področij. Povrh, odprti bralni predelni imajo bolj stabilno smerno RNA/DNA dvojno vijačnico, kot potencialno ujemajoč protismeren dupleks. Raziskava temelji na izračunih, koliko proste energije je potrebno, da se dvojna vijačnica razvije. Več energije, kot je potrebno stabilnejša je struktura. Rezultati torej prikazujejo, da so geni stabilnejši od medgenskih področij. Torej lahko povzamemo, da stabilnost DNA/DNA in RNA/DNA dupleksa vpliva na mRNA prepis.

== Dominik Kert: Kako osteokalcin vpliva na reprodukcijo organizmov ==
Nove raziskave kažejo povezave med reprodukcijo in skeletnim sistemom. Kosti so sestavljene iz osteoblastov. Produkt le teh je hormon osteokalcin. Ta hormon pa zelo vpliva na produkcijo testosterona, ampak ne neposredno. Deluje namreč na leydigove celice, ki se nahajajo med tubuli v testisih. Raziskovalci so odkrivali te rezultate tako, da bo samcem miši vbrizgali osteokalcin-takrat se je raven testosterona dvignila. Po drugi strani pa so miškam odstranili gen, ki kodira ta hormon. Prišlo je do osupljivih rezultatov. Mišim se je zmanjšala plodnost, in sicer tako, da se je zmanjšala količina sperme, zmanjšala število zdravih spermijev in veliko jih je tudi pomrlo že v modih. Dokazali so tudi veliko signalnih poti med reproduciranjem in okostjem. In sicer če organizem dobro funkcionira in ima dovolj zalog hrane se je tudi sposoben razmnoževati. In pa tudi povezavo med debelostjo in neplodnostjo. Ampak to še ni vse. Mišim se je tudi spremenilo partnersko vedenje: manjkrat so postavljali gnezda. To je bila posledica nabiranja luteinizirajočega hormona. Poleg slednjega hormona se je začela pojavljati tudi večja količina estrogena. Te dve posledici pa lahko povežemo s staranjem moškega, ko testosteron začne vpadati, estrogen in luteinizirajoči hormon pa narasteta. Zaradi teh ugotovitev bi bilo lahko v prihodnje možno zdravljenje neplodnosti pri ljudeh.

== Živa Brglez: Kompleks Mre11 ==
Med podvojevanjem dvojne vijačnice DNA včasih pride do napak, ki jih je nujno potrebno popraviti, oziroma obnoviti v njeno izvirno obliko, če je prišlo do poškodbe zaradi mutagenih dejavnikov iz okolja, da se ne prenesejo naprej v hčerinske celice in prihodnje rodove. Za to skrbijo raznovrstni popravljalni mehanizmi. V primeru dvojnega preloma verige DNA (Double-Strand Breaks – DBS) je kompleks Mre11, sestavljen iz treh različnih proteinov: dimera mejotske rekombinacije 11 (Mre11), dimera Rad50 in Nbs1, ključnega pomena za celični odziv na poškodbe DNA. Mre11 in Nbs1 skupaj z delom Rad50 tvorita glavno domeno kompleksa, iz katere izraščajo obvite vijačnice (coiled-coil) Rad50. Poznana sta dva načina poprave preloma dvojne verige DNA, homologna rekombinacija (HR) in sklepanje nehomolognih koncev verig DNA (NHEJ). Kompleks Mre11 sodeluje pri obeh, strukturno in encimatsko. Strukturno tako, da glavno domeno veže poškodovan del DNA, medtem ko se obviti vijačnici povežeta z obvitima vijačnicama drugega kompleksa Mre11 in držita skupaj prelomljene verige. Encimatsko pa s spodbujanjem odstranitve koncev in posredovanjem informacije ATM (ataxia-telengastia-mutated), katerega del se fosforilira na glavni domeni kompleksa Mre11 in tako vpokliče še ostale molekule odgovorne za popravo. Kompleks Mre11 skrbi tudi za homeostazo telomerov z regulira njihove dolžine, in posredno za razvoj imunskega sistema. V primeru mutacije v katerem izmed genov, ki kodirajo gradbene proteine kompleksa Mre11, pride do različnih dednih bolezni, ki se kažejo fenotipsko podobno – značilni sta hiperobčutljivost na radiacijo in mikrocefalija.

== Taja Karner: Alkohol omogoča lažje nalaganje CD1d molekul, s tem aktivira NKT celice in zmanjša pojavljanje sladkorne bolezni pri NOD miškah ==
V raziskavi, ki sem si jo izbrala raziskujejo pozitivne učinke alkohola. Vse več raziskav kaže, da zmerno uživanje alkoholnih pijač pripomore k boljšemu delovanju našega mehanizma. V tej raziskavi pa jih je zanimalo predvsem kakšen je vpliv alkohola na prirojen imunski sistem. Raziskave so potekale in vitro z uporabo α-GalCer molekul in in vivo na NOD miših. NKT celice, ki jih omenjam v svoji seminarski, pa so heterogene skupine, sestavljene iz NK celic in T celic. Te celice prepoznavajo antigene CD1d, ki se izražajo na površini antigen-predstavitvenih celic. To so celice, ki imajo sposobnost preoblikovanja antigenov, tako da jih T-celice prepoznajo in ustvarijo ustrezen odgovor. Ugotovili so, da alkohol izboljša nalaganje CD1d molekul, s tem aktivacijo NKT celic in tako zmanjša možnost za razvoj diabetesa. Pri NOD miših, ki so jim dajali 5 % alkohola se je pokazalo zmanjšano število diabetesa in manjša koncentracija glukoze ob testiranju. Zanimivo je, da te miši niso kazale nikakršnih znakov alkoholizma. Pri ljudeh bi takšna koncentracija, preračunana glede na maso človeka, povzročila visoko stopnjo opojnost. Kljub temu pa je bila v mišji krvi razmeroma mala količina alkohola, kar kaže na bistveno večjo zmožnost razstrupljanja alkohola pri NOD miših, kot jo imamo ljudje.

== Karmen Gec: Učinki vadbe in/ali dodajanja antioksidantov na gene endotelnih celic ==
Teoretično je raziskano, da so antioksidanti v sadju in zelenjavi pomembni pri zaviraju oksidativnih mehanizmov, ki vodijo do različnih degenerativnih bolezni, tudi srčno-žilnih bolezni. V obravnavani raziskavi avtorji na podganah ugotavljajo različno izražanje genov endotelnih celic glede na dodatek antioksidantov, glede na vadbo ali kombinacijo obojega. Navajajo, da je pri ednoteliju zelo pomembno, da razlikujemo med fiziološkimi in patološkimi t.i. Reaktivnimi kisikovimi zvrstmi (v nadaljevanju RKZ). Izražanje genov endotelnih celic so ugotavljali v področju srčnega endotelija (levi ventrikel) in žilnega endotelija (koronarna arterija). V raziskavi so ugotovili, da je gen RhoA, ki je pomemben pri srčno-žilnih boleznih kazal znižan učinek pri vadbi ter povišan pri dodatku antioksidantov v področju levega ventrikla. Poleg tega pa je še IL-6, pomemben gen pri vnetju, znižal učinek pri vseh treh dodanih tretmajih. Tako izražanje obeh genov z dodajanjem vadbe in/ali antioksidantov poda vpogled v molekulske mehanizme srčnožilne bolezni.

== Tjaša Goričan: Molekulske tarče oksidativnega stresa ==
Aerobni organizmi so življensko odvisni od procesov celičnega dihanja, pri čemer pa vedno nastajajo za biološke makromolekule (lipide, DNA, proteine) škodljivi kisikovi radikali. Organizmi so razvili obrambne mehanizme, ki preprečujejo potencialno škodo. Pri tem se ohranja neko ravnovesje med kisikovimi radikali in antioksidanti, katere mora organizem pridobiti s hrano. Antioksidanti (vitamini C, E, koencim Q10, karotenoidi itd.) so snovi, ki delujejo kot katalizatorji in celice varujejo pred oksidacijo. Porušeno ravnotežje povzroči oksidativni stres, posledice katerega so lahko različne bolezni (Alzheimerjeva bolezen, Parkinsonova bolezen, rak, itd.)in staranje. Zmerna oksidacija sproži apoptozo (programirano celično smrt), hujši in intenzivni oksidativni stres pa lahko povzroči celično smrt in celo nekrozo (odmrtje celic/ tkiva). V mojem članku so poskuse izvajali na bakterijah in kvasovkah ter ugotovili, da so pri obeh prvotne tarče ROS (reaktivne spojine, ki vsebujejo atom kisika) različne. Rezultati: pri prokariontih je DNA prvotna tarča, pri evkariontih pa ne. Vzrok za to je različen prag občutljivosti molekul pri različnih organizmih. Identifikacija primarnih tarč oksidativnega stresa bi odprla nove možnosti za terapije bolezni povezane z njim.

== Jernej Mustar: Odpornost srpastih celic na okužbo z plazmodijem ==
V mojem članku so raziskovali fenomen, ki se pojavlja pri obolelih za anemijo srpastih celic(HbS homozigotni) in HbS heterozigotnih, in sicer toleranca do okužbe z Plasmodijem. V raziskavi so uporabili Plasmodium berghei, ki je modelni organizem za razumevanje človeške malarije. Ta povzroča t.i. možgansko malarijo (experimental cerebral malaria-ECM). Znanstveniki so prišli do zelo zanimivih odkritij. Ugotovili so namreč, da se glavni vzrok imunosti skriva v nalaganju nizkih količin prostega hema v krvi in povečane ekspresije stresno-odgovornega encima HemOxigenaze1(ki razgrajuje prosti hem). Pri katalizi hema nastaja CO, ki se veže na hemoglobin in prepreči odcepitev prostetične skupine (hema), saj je le ta glavni vzrok patogeneze ECM. Ta spoznanja so, po mojem mnenju, človeštvo privedla korak bližje k odkritju zdravila za malarijo.
== Ula Štok: Mutacija mitohondrijske DNA v povezavi z rakom debelega črevesa kot posledica abnormalnega delovanja citokroma c oksidaze ==
Mutaciji Ala501Pro in Gly171Asp pri bakteriji ''Rhodobacter sphaerodies'', ki sovpadata z mutacijama Gly125Asp ter Ser458Pro v človeškem telesu povzročata nastajanje rakavih celic, ki vodi do razvoja raka debelega črevesa. Gre za mutaciji v podenoti I kompleksa IV dihalne verige. Procesi dihalne verige so izrednega pomena za proizvodnjo energije, ki jo celica potrebuje za nemoteno delovanje. Elektroni, ki so višek procesa glikolize, se namreč shranjujejo na t.i kofaktorjih in porabljajo kot gonilna sila dihalne verige v mitohondriju. Kompleks IV je predzadnji v tej verigi in je generalno gledano sestavljen iz 13 podenot. Zgradba in sestava vseh podenot še vedno ni povsem jasna/raziskana (predvsem tistih manjših, do 90 aminokislinskih ostankov). Mutacija na podenoti I totalno onemogoči delovanje celotnega sistema. Gre namreč za oviran in predvsem upočasnjen prenos elektronov iz posameznih regij, kar ima za posledico tudi manjšo aktivnost prenosa protonov v intermembranski prostor mitohondrija. Na bolezenski ravni to povzroči nastajanje rakastih celic iz dveh razlogov. Pride do nastanka ROS (Reactive oxygen species), ki so za celico strupeni (v prekomernih količinah) ter do porušenega Δψ / ΔpH razmerja. Sprememba Δψ / ΔpH razmerja povzroči, da se zmanjša transport Ca2+ ionov v mitohondrij, kar ovira delovanje le-tega, saj so Ca2+ ioni namreč odgovorni za regulacijo procesov dihalne verige.

BIO1-seminar 2011

2011-03-16T16:14:46Z

MatjaZalar: /* Seznam seminarjev */

= Temelji biokemije- seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsak ponedeljek od 10:00 do 11:30.

Ocena seminarjev predstavlja ??% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev ==
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;"
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Slovenski naslov članka'''
| align="center" style="background:#f0f0f0;"|'''Faktor vpliva revije'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 2'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 3'''
|-
| BOTONJIĆ SANDI||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Sandi_Botonji.C4.87:_Tioredoksinu_podoben_protein_.28TXNL2.29_.C5.A1.C4.8Diti_kancerogene_celice_pred_oksidativnim_stresom Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom]
||15.387||28.02.||03.03.||07.03.||RODE URŠKA||KERIN INES||OGRIS IZA
|-
| VRANKAR ANDREJ||Število lasno-mešičnih matičnih celic se v plešastem lasišču moških z androgeno alopecijo ohranja za razliko od števila CD200-rich in CD34-positive lasno-mešičnih predniških celic||||28.02.||03.03.||07.03.||HROVAT KARMEN||BOHNEC IVO||JAVORŠEK KAJA
|-
| ZALAR MATJA||Protein p53||||28.02.||03.03.||07.03.||OGRIS IZA||CRČEK MITJA||ZOTTEL ALJA
|-
| ZOTTEL ALJA||Vloga imunskega sistema pri aterosklerozi||31.434||07.03.||10.03.||14.03.||RADOJKOVIĆ MARKO||KERT DOMINIK||HROVAT KARMEN
|-
| DOLINAR ANA||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Ana_Dolinar:_Prilagojena_ali_prilagodljiva_imunost.3F_Primer_naravnih_celic_ubijalk Prirojena ali prilagodljiva imunost? Primer naravnih celic ubijalk]||28||07.03.||10.03.||14.03.||RAUTER URŠKA||MOHAR MAŠA||VERBANČIČ JANA
|-
| RAUTER URŠKA||Razvojna vloga Srf, kortikalnega citoskeleta in celične oblike v orientaciji epidermalnega vretena||19.527||07.03.||10.03.||14.03.||MUSTAR JERNEJ||JAVORŠEK KAJA||MOHAR MAŠA
|-
| MOHAR MAŠA||Sladkorna bolezen tipa 2 kot bolezen imunskega sistema||30,006||14.03.||17.03.||21.03.||VENE ROK||RAUTER URŠKA||GORIČAN TJAŠA
|-
| POHLEVEN ŠPELA||Prioni||34||14.03.||17.03.||21.03.||KEPIC LEA||RADOJKOVIĆ MARKO||DOLINAR ANA
|-
| KEPIC LEA||Agonisti adrenoreceptorjev β2||34.48||14.03.||17.03.||21.03.||VRANKAR ANDREJ||BRATOVŠ ANDREJA||MUSTAR JERNEJ
|-
| KMETIČ MIRJAM||Celična regulacija metabolizma železa||5,371||14.03.||17.03.||21.03.||MARIĆ TAMARA||REMŠKAR MAJA||KOMAN KATRA
|-
| JARC VERONIKA||Eksperimentalni modeli za študijo imunobiologije hepatitisa C||3.26||14.03.||21.03.||28.03.||REMŠKAR MAJA||MUSTAR JERNEJ||KEPIC LEA
|-
| KOMAN KATRA||naslov||||16.03.||21.03.||28.03.||ČUPOVIĆ VANA||KARNER TAJA||KMETIČ MIRJAM
|-
| OGRIS IZA||Zakaj imajo možgani glikogen?||5,125||14.03.||21.03.||28.03.||KNAPIČ EVA||BRGLEZ ŽIVA||VRANKAR ANDREJ
|-
| KERIN INES||Kanabinoidi za zdravljenje shizofrenije? Uravnotežena nevrokemična sestava za škodljive in terapevtske učinke uživanja konoplje||4.458||14.03.||21.03.||28.03.||ŠTOK ULA||ŠTEMBERGER ROK||KERT DOMINIK
|-
| VERBANČIČ JANA||Apoptozi podobna smrt v bakterijah, ki jo povzroča HAMLET, človeški mlečni lipidno-proteinski kompleks||4.351||21.03.||28.03.||04.04.||KARNER TAJA||ZOTTEL ALJA||KNAPIČ EVA
|-
| KNAPIČ EVA||Kako virusi vodijo delovanje celice.||14.101||21.03.||28.03.||04.04.||ZALAR MATJA||POHLEVEN ŠPELA||LORBEK SARA
|-
| REMŽGAR ANA||naslov||||21.03.||28.03.||04.04.||BOTONJIĆ SANDI||LORBEK SARA||ČUPOVIĆ VANA
|-
| GRDADOLNIK MAJA||naslov||||21.03.||28.03.||04.04.||MOHAR MAŠA||REMŽGAR ANA||FRANKO NIK
|-
| JAVORŠEK KAJA||Potencial matične celice pri Parkinsonovi bolezni in molekularni faktorji za tvorbo dopaminergičnih nevronov||4.139||28.03.||04.04.||11.04.||GEC KARMEN||MARIĆ TAMARA||RADOJKOVIĆ MARKO
|-
| BRATOVŠ ANDREJA||Vloga GPCR v patologiji Alzheimerjeve bolezni||26||28.03.||04.04.||11.04.||ZOTTEL ALJA||ČUPOVIĆ VANA||GRDADOLNIK MAJA
|-
| CRČEK MITJA||Matične celice||7.365||28.03.||04.04.||11.04.||BOHNEC IVO||KMETIČ MIRJAM||BRATOVŠ ANDREJA
|-
| MARIĆ TAMARA||ciljanje kemokinskih receptorjev ob alergijskih obolenjih||5.155||28.03.||04.04.||11.04.||NAVODNIK URŠKA||GEC KARMEN||REMŠKAR MAJA
|-
| ŠTEMBERGER ROK||povečano izražanje in imunogenosti HIV proteinov po inaktivaciji encimske aktivnosti||||04.04.||11.04.||18.04.||JAVORŠEK KAJA||VRANKAR ANDREJ||BOTONJIĆ SANDI
|-
| LORBEK SARA||Interakcije maščobna kislina - gen, adipokini in debelost||3.072||04.04.||11.04.||18.04.||POHLEVEN ŠPELA||KNAPIČ EVA||VENE ROK
|-
| REMŠKAR MAJA||naslov||||04.04.||11.04.||18.04.||KERIN INES||POVŠE KATJA||CRČEK MITJA
|-
| ČUPOVIĆ VANA||naslov||||04.04.||11.04.||18.04.||REMŽGAR ANA||VERBANČIČ JANA||RODE URŠKA
|-
| RODE URŠKA||naslov||||24.04.||03.05.||09.05.||GRDADOLNIK MAJA||FRANKO NIK||MARIĆ TAMARA
|-
| RADOJKOVIĆ MARKO||naslov||||24.04.||03.05.||09.05.||FRANKO NIK||VENE ROK||POVŠE KATJA
|-
| VENE ROK||naslov||||24.04.||03.05.||09.05.||VERBANČIČ JANA||NAVODNIK URŠKA||ZALAR MATJA
|-
| FRANKO NIK||naslov||||24.04.||03.05.||09.05.||ŠTEMBERGER ROK||HROVAT KARMEN||BOHNEC IVO
|-
| HROVAT KARMEN||naslov||||04.05.||09.05.||16.05.||KERT DOMINIK||JARC VERONIKA||KARNER TAJA
|-
| AMBROŽIČ MATEVŽ||naslov||||04.05.||09.05.||16.05.||LORBEK SARA||KEPIC LEA||REMŽGAR ANA
|-
| NAVODNIK URŠKA||naslov||||04.05.||09.05.||16.05.||AMBROŽIČ MATEVŽ||ŠTOK ULA||ŠTEMBERGER ROK
|-
| BRGLEZ ŽIVA||naslov||||09.05.||16.05.||23.05.||DOLINAR ANA||BOTONJIĆ SANDI||JARC VERONIKA
|-
| KARNER TAJA||naslov||||09.05.||16.05.||23.05.||KOMAN KATRA||OGRIS IZA||NAVODNIK URŠKA
|-
| KERT DOMINIK||naslov||||09.05.||16.05.||23.05.||GORIČAN TJAŠA||GRDADOLNIK MAJA||RAUTER URŠKA
|-
| MUSTAR JERNEJ||naslov||||16.05.||23.05.||30.05.||JARC VERONIKA||AMBROŽIČ MATEVŽ||BRGLEZ ŽIVA
|-
| GEC KARMEN||naslov||||16.05.||23.05.||30.05.||POVŠE KATJA||ZALAR MATJA||AMBROŽIČ MATEVŽ
|-
| GORIČAN TJAŠA||naslov||||16.05.||23.05.||30.05.||KMETIČ MIRJAM||RODE URŠKA||POHLEVEN ŠPELA
|-
| BOHNEC IVO||naslov||||23.05.||30.05.||06.06.||CRČEK MITJA||GORIČAN TJAŠA||ŠTOK ULA
|-
| ŠTOK ULA||Mutacija mitohondrijske DNA v povezavi z rakom debelega črevesa kot posledica abnormalnega delovanja citokroma c oksidaze||||23.05.||30.05.||06.06.||BRGLEZ ŽIVA||DOLINAR ANA||KERIN INES
|-
| nihce ||naslov||||23.05.||30.05.||06.06.||BRATOVŠ ANDREJA||KOMAN KATRA||GEC KARMEN
|}

==Naloga==
* samostojno pripraviti seminar, katerega osnova je znanstveni članek s področja biokemije, ki ga po želji izberete v reviji s področja biokemije, ki ima faktor vpliva večji kot 3 in je bil objavljen v letu 2011. Poleg tega članka morate za seminar uporabiti še najmanj pet drugih virov! http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR
* osnovni članek in naslov pošljete meni, najkasneje pet dni pred rokom za oddajo (rok-5), da ocenim, če je primeren za predstavitev. Naslov vpišete v tabelo, takoj ko ste si ga izbrali!
* [[BIO1 Povzetki seminarjev|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom. Povezave do slik so dobrodošle, niso pa nujne.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 1800 do 2000 besed), vsebovati mora najmanj eno sliko. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Natisnjen seminar oddajte do roka vsakemu od recenzentov (docentu ga pošljite po e-pošti v formatu .doc ali .docx).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela, v predpisanem formatu elektronskega obrazca na internetu.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 15 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava- 5 minut. Recenzenti podajo oceno predstavitve in postavijo vsak vsaj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek na wikiju morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [[https://spreadsheets.google.com/viewform?formkey=dFM2SktfM3Q4VU1wNUQzdU45OTlWVXc6MA recenzentsko poročilo]] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://spreadsheets.google.com/viewform?formkey=dFd3TGhLV3ZSa2xsLVlmMVVUaEFURWc6MA mnenje] najkasneje v treh dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Faktor vpliva==
Faktor vpliva (angl. impact factor) neke revije pove, kolikokrat so bili v poprečju citirani članki v tej reviji v dveh letih skupaj pred objavo tega faktorja. Faktorje vpliva za posamezno revijo lahko najdete v [http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR COBISS-u]. V polje "Naslov revije" vnesite ime revije za katero želite izvedeti faktor vpliva in pritisnite na gumb POIŠČI. V skrajnem desnem stolpcu se bodo izpisali faktorji vpliva za revije, ki ustrezajo vašim iskalnim kriterijem. Zadetkov za posamezno revijo je več zato, ker so navedeni faktorji vpliva za posamezno leto. Za leto 2011 faktorji vpliva še niso objavljeni, zato se orientirajte po faktorjih vpliva zadnjih par let. Če faktorja vpliva za vašo izbrano revijo ne najdete v bazi COBISS, potem izberite članek iz kakšne druge revije.

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.google.com/url?sa=t&source=web&cd=6&sqi=2&ved=0CEUQFjAF&url=http%3A%2F%2Fwww.tre.sik.si%2Fmain%2Fpomoc%2Ffiles%2Fcitiranje_in_navajanje_virov.pdf&rct=j&q=citiranje%20po%20pravilniku%20ISO%20690&ei=jPBqTe6FC9DKswaWk-TmDA&usg=AFQjCNF8r6X9Y781sanDObaXNdCew4suUg&sig2=cTqKObSJsTicekWGRGa72g&cad=rja Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO1 Povzetki seminarjev 2011

2011-02-28T22:46:22Z

MatjaZalar: /* Ime in priimek: Naslov sdčkfjlsadjfkljsad fjasd test testklsnklaf sdlk fglkasdjklg */

== Matja Zalar: Protein p53 ==
Protein p53, včasih imenovan tudi varuh genoma, kodira gen TP53 na sedemnajstem kromosomu. Je eden izmed tako imenovanih tumor-supresorskih proteinov, ki, kot to sporoča že samo ime, zavirajo nastanek in rast tumorjev. Na področju razumevanja delovanja, vloge in strukture proteina p53 in njegovih mutantov se izvaja veliko raziskav. Trenutno je p53 najbolj raziskan tumor-supresorski protein, še zdaleč pa ni edini. Gre za protein, ki se kopiči v jedru in z vezavo na DNA v obliki teramera nadzoruje in regulira procese kot so apoptoza, zaustavitev celičnega cikla in popravljanje poškodovane DNA. Za raziskovalce je še posebno zanimiv zaradi dejstva, da v nemutirani obliki zavira nastanek in rast tumojev, njegove GOF mutirane oblike pa pripomorejo k nenadzorovani delitvi celic in nastanku rakastih tkiv. Veliko raziskav se ukvarjaja z iskanjem snovi, ki bi obnovile osnovno obliko p53, oziroma uničile mutantske oblike p53 v rakastih celicah ter s tem uničile tumor. To pa bi lahko bistveno izboljšalo tehnike zdravljenja rakavih obolenj in odziv človeškega organizma na ta zdravljenja. Odkrili so že kar nekaj takšnih snovi (RITA, PRIMA, nutlin3), ki pa jih še vedno testirajo in še niso v redni uporabi pri zdravljenju rakavih obolenj.

== Ime in priimek: Naslov mojega seminarja ==
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam vel gravida urna. Nunc dignissim, augue eu pharetra volutpat, nisi neque mattis leo, sed rhoncus sem purus eget est. Etiam bibendum mi sit amet augue volutpat viverra. Sed ac nibh eu risus pellentesque commodo eget non odio. Vivamus nec odio vel felis tristique ultricies. Morbi sed mauris non est congue adipiscing. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean at ante ut arcu pretium mollis. Ut quis quam ut lacus auctor auctor. Pellentesque lobortis sagittis dolor ac gravida. Nulla in tellus dolor, a malesuada risus. Suspendisse ornare, mi in molestie gravida, velit sapien dapibus mauris, id ultrices velit libero id sapien. Sed pharetra dictum lectus in egestas.
Phasellus tempor, arcu a venenatis faucibus, orci arcu imperdiet mauris, quis adipiscing quam lectus vel dolor. Vestibulum sagittis ante quis ligula ullamcorper eget convallis justo fringilla. Mauris eget tellus at ante vulputate fermentum. Mauris placerat, arcu eu lobortis facilisis, neque dui pellentesque sapien, sit amet rutrum neque nisl vitae turpis. Donec urna elit, imperdiet nec tempus in, lacinia vitae ligula. Integer commodo, dolor non semper egestas, magna ipsum imperdiet quam, a lobortis purus elit bibendum mi. Cras in tortor non mauris pulvinar egestas.

== Ime in priimek: Naslov mojega seminarja ==
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam vel gravida urna. Nunc dignissim, augue eu pharetra volutpat, nisi neque mattis leo, sed rhoncus sem purus eget est. Etiam bibendum mi sit amet augue volutpat viverra. Sed ac nibh eu risus pellentesque commodo eget non odio. Vivamus nec odio vel felis tristique ultricies. Morbi sed mauris non est congue adipiscing. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean at ante ut arcu pretium mollis. Ut quis quam ut lacus auctor auctor. Pellentesque lobortis sagittis dolor ac gravida. Nulla in tellus dolor, a malesuada risus. Suspendisse ornare, mi in molestie gravida, velit sapien dapibus mauris, id ultrices velit libero id sapien. Sed pharetra dictum lectus in egestas.
Phasellus tempor, arcu a venenatis faucibus, orci arcu imperdiet mauris, quis adipiscing quam lectus vel dolor. Vestibulum sagittis ante quis ligula ullamcorper eget convallis justo fringilla. Mauris eget tellus at ante vulputate fermentum. Mauris placerat, arcu eu lobortis facilisis, neque dui pellentesque sapien, sit amet rutrum neque nisl vitae turpis. Donec urna elit, imperdiet nec tempus in, lacinia vitae ligula. Integer commodo, dolor non semper egestas, magna ipsum imperdiet quam, a lobortis purus elit bibendum mi. Cras in tortor non mauris pulvinar egestas.

BIO1-seminar 2011

2011-02-28T09:55:10Z

MatjaZalar: /* Seznam seminarjev */

= Temelji biokemije- seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsak ponedeljek od 10:00 do 11:30.

Ocena seminarjev predstavlja ??% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev ==
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;"
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Slovenski naslov članka'''
| align="center" style="background:#f0f0f0;"|'''Faktor vpliva revije'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 2'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 3'''
|-
| BOTONJIĆ SANDI||Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom.||||28.02.||03.03.||07.03.||RODE URŠKA||KERIN INES||OGRIS IZA
|-
| VRANKAR ANDREJ||Število lasno-mešičnih matičnih celic se v plešastem lasišču moških z androgeno alopecijo ohranja za razliko od števila CD200-rich in CD34-positive lasno-mešičnih predniških celic||||28.02.||03.03.||07.03.||HROVAT KARMEN||BOHNEC IVO||JAVORŠEK KAJA
|-
| ZALAR MATJA||Protein p53||||28.02.||03.03.||07.03.||OGRIS IZA||CRČEK MITJA||ZOTTEL ALJA
|-
| ZOTTEL ALJA||Vloga imunskega sistema pri aterosklerozi||||07.03.||10.03.||14.03.||RADOJKOVIĆ MARKO||KERT DOMINIK||HROVAT KARMEN
|-
| DOLINAR ANA||Celice ubijalke||||07.03.||10.03.||14.03.||RAUTER URŠKA||MOHAR MAŠA||VERBANČIČ JANA
|-
| RAUTER URŠKA||naslov svojega seminarja||||07.03.||10.03.||14.03.||MUSTAR JERNEJ||JAVORŠEK KAJA||MOHAR MAŠA
|-
| MOHAR MAŠA||naslov||||14.03.||17.03.||21.03.||VENE ROK||RAUTER URŠKA||GORIČAN TJAŠA
|-
| POHLEVEN ŠPELA||jhfjsdf sdh fjhds v||12||14.03.||17.03.||21.03.||KEPIC LEA||RADOJKOVIĆ MARKO||DOLINAR ANA
|-
| KEPIC LEA||naslov||||14.03.||17.03.||21.03.||VRANKAR ANDREJ||BRATOVŠ ANDREJA||MUSTAR JERNEJ
|-
| KMETIČ MIRJAM||naslov||||14.03.||17.03.||21.03.||MARIĆ TAMARA||REMŠKAR MAJA||KOMAN KATRA
|-
| JARC VERONIKA||naslov||||14.03.||21.03.||28.03.||REMŠKAR MAJA||MUSTAR JERNEJ||KEPIC LEA
|-
| KOMAN KATRA||naslov||||14.03.||21.03.||28.03.||ČUPOVIĆ VANA||KARNER TAJA||KMETIČ MIRJAM
|-
| OGRIS IZA||naslov||||14.03.||21.03.||28.03.||KNAPIČ EVA||BRGLEZ ŽIVA||VRANKAR ANDREJ
|-
| KERIN INES||naslov||||14.03.||21.03.||28.03.||KARNER TAJA||ŠTEMBERGER ROK||KERT DOMINIK
|-
| VERBANČIČ JANA||naslov||||21.03.||28.03.||04.04.||ŠTOK ULA||ZOTTEL ALJA||KNAPIČ EVA
|-
| KNAPIČ EVA||naslov||||21.03.||28.03.||04.04.||ZALAR MATJA||POHLEVEN ŠPELA||LORBEK SARA
|-
| REMŽGAR ANA||naslov||||21.03.||28.03.||04.04.||BOTONJIĆ SANDI||LORBEK SARA||ČUPOVIĆ VANA
|-
| GRDADOLNIK MAJA||naslov||||21.03.||28.03.||04.04.||MOHAR MAŠA||REMŽGAR ANA||FRANKO NIK
|-
| JAVORŠEK KAJA||naslov||||28.03.||04.04.||11.04.||GEC KARMEN||MARIĆ TAMARA||RADOJKOVIĆ MARKO
|-
| BRATOVŠ ANDREJA||naslov||||28.03.||04.04.||11.04.||ZOTTEL ALJA||ČUPOVIĆ VANA||GRDADOLNIK MAJA
|-
| CRČEK MITJA||naslov||||28.03.||04.04.||11.04.||BOHNEC IVO||KMETIČ MIRJAM||BRATOVŠ ANDREJA
|-
| MARIĆ TAMARA||naslov||||28.03.||04.04.||11.04.||NAVODNIK URŠKA||GEC KARMEN||REMŠKAR MAJA
|-
| ŠTEMBERGER ROK||naslov||||04.04.||11.04.||18.04.||JAVORŠEK KAJA||VRANKAR ANDREJ||BOTONJIĆ SANDI
|-
| LORBEK SARA||naslov||||04.04.||11.04.||18.04.||POHLEVEN ŠPELA||KNAPIČ EVA||VENE ROK
|-
| REMŠKAR MAJA||naslov||||04.04.||11.04.||18.04.||KERIN INES||POVŠE KATJA||CRČEK MITJA
|-
| KARNER TAJA||naslov||||04.04.||11.04.||18.04.||REMŽGAR ANA||VERBANČIČ JANA||RODE URŠKA
|-
| RODE URŠKA||naslov||||24.04.||03.05.||09.05.||GRDADOLNIK MAJA||FRANKO NIK||MARIĆ TAMARA
|-
| RADOJKOVIĆ MARKO||naslov||||24.04.||03.05.||09.05.||FRANKO NIK||VENE ROK||POVŠE KATJA
|-
| VENE ROK||naslov||||24.04.||03.05.||09.05.||VERBANČIČ JANA||NAVODNIK URŠKA||ZALAR MATJA
|-
| AMBROŽIČ MATEVŽ||naslov||||24.04.||03.05.||09.05.||ŠTEMBERGER ROK||HROVAT KARMEN||BOHNEC IVO
|-
| HROVAT KARMEN||naslov||||04.05.||09.05.||16.05.||KERT DOMINIK||JARC VERONIKA||KARNER TAJA
|-
| FRANKO NIK||naslov||||04.05.||09.05.||16.05.||LORBEK SARA||KEPIC LEA||REMŽGAR ANA
|-
| NAVODNIK URŠKA||naslov||||04.05.||09.05.||16.05.||AMBROŽIČ MATEVŽ||ŠTOK ULA||ŠTEMBERGER ROK
|-
| BRGLEZ ŽIVA||naslov||||09.05.||16.05.||23.05.||DOLINAR ANA||BOTONJIĆ SANDI||JARC VERONIKA
|-
| ČUPOVIĆ VANA||naslov||||09.05.||16.05.||23.05.||KOMAN KATRA||OGRIS IZA||NAVODNIK URŠKA
|-
| KERT DOMINIK||naslov||||09.05.||16.05.||23.05.||GORIČAN TJAŠA||GRDADOLNIK MAJA||RAUTER URŠKA
|-
| POVŠE KATJA||naslov||||16.05.||23.05.||30.05.||JARC VERONIKA||AMBROŽIČ MATEVŽ||BRGLEZ ŽIVA
|-
| GEC KARMEN||naslov||||16.05.||23.05.||30.05.||POVŠE KATJA||ZALAR MATJA||AMBROŽIČ MATEVŽ
|-
| GORIČAN TJAŠA||naslov||||16.05.||23.05.||30.05.||KMETIČ MIRJAM||RODE URŠKA||POHLEVEN ŠPELA
|-
| BOHNEC IVO||naslov||||23.05.||30.05.||06.06.||CRČEK MITJA||GORIČAN TJAŠA||ŠTOK ULA
|-
| ŠTOK ULA||naslov||||23.05.||30.05.||06.06.||BRGLEZ ŽIVA||DOLINAR ANA||KERIN INES
|-
| MUSTAR JERNEJ||naslov||||23.05.||30.05.||06.06.||BRATOVŠ ANDREJA||KOMAN KATRA||GEC KARMEN
|}

==Naloga==
* samostojno pripraviti seminar, katerega osnova je znanstveni članek s področja biokemije, ki ga po želji izberete v reviji s področja biokemije, ki ima faktor vpliva večji kot 3 in je bil objavljen v letu 2011. Poleg tega članka morate za seminar uporabiti še najmanj pet drugih virov! http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR
* osnovni članek in naslov pošljete meni, najkasneje pet dni pred rokom za oddajo (rok-5), da ocenim, če je primeren za predstavitev. Naslov vpišete v tabelo, takoj ko ste si ga izbrali!
* [[BIO1 Povzetki seminarjev|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom. Povezave do slik so dobrodošle, niso pa nujne.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 1800 do 2000 besed), vsebovati mora najmanj eno sliko. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Natisnjen seminar oddajte do roka vsakemu od recenzentov (docentu ga pošljite po e-pošti v formatu .doc ali .docx).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela, v predpisanem formatu elektronskega obrazca na internetu.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 15 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava- 5 minut. Recenzenti podajo oceno predstavitve in postavijo vsak vsaj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek na wikiju morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [[https://spreadsheets.google.com/viewform?formkey=dFM2SktfM3Q4VU1wNUQzdU45OTlWVXc6MA recenzentsko poročilo]] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://spreadsheets.google.com/viewform?formkey=dFd3TGhLV3ZSa2xsLVlmMVVUaEFURWc6MA mnenje] najkasneje v treh dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.google.com/url?sa=t&source=web&cd=6&sqi=2&ved=0CEUQFjAF&url=http%3A%2F%2Fwww.tre.sik.si%2Fmain%2Fpomoc%2Ffiles%2Fcitiranje_in_navajanje_virov.pdf&rct=j&q=citiranje%20po%20pravilniku%20ISO%20690&ei=jPBqTe6FC9DKswaWk-TmDA&usg=AFQjCNF8r6X9Y781sanDObaXNdCew4suUg&sig2=cTqKObSJsTicekWGRGa72g&cad=rja Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO1-seminar 2011

2011-02-28T09:53:46Z

MatjaZalar: /* Seznam seminarjev */

= Temelji biokemije- seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsak ponedeljek od 10:00 do 11:30.

Ocena seminarjev predstavlja ??% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev ==
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;"
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Slovenski naslov članka'''
| align="center" style="background:#f0f0f0;"|'''Faktor vpliva revije'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 2'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 3'''
|-
| BOTONJIĆ SANDI||Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom.||||28.02.||03.03.||07.03.||RODE URŠKA||KERIN INES||OGRIS IZA
|-
| VRANKAR ANDREJ||Število lasno-mešičnih matičnih celic se v plešastem lasišču moških z androgeno alopecijo ohranja za razliko od števila CD200-rich in CD34-positive lasno-mešičnih predniških celic||||28.02.||03.03.||07.03.||HROVAT KARMEN||BOHNEC IVO||JAVORŠEK KAJA
|-
| ZALAR MATJA||Protein p53||||28.02.||03.03.||07.03.||OGRIS IZA||CRČEK MITJA||ZOTTEL ALJA
|-
| ZOTTEL ALJA||Vloga imunskega sistema pri aterosklerozi||||07.03.||10.03.||14.03.||RADOJKOVIĆ MARKO||KERT DOMINIK||HROVAT KARMEN
|-
| DOLINAR ANA||Celice ubijalke||||07.03.||10.03.||14.03.||RAUTER URŠKA||MOHAR MAŠA||VERBANČIČ JANA
|-
| RAUTER URŠKA||naslov svojega seminarja||||07.03.||10.03.||14.03.||MUSTAR JERNEJ||JAVORŠEK KAJA||MOHAR MAŠA
|-
| MOHAR MAŠA||naslov||||14.03.||17.03.||21.03.||VENE ROK||RAUTER URŠKA||GORIČAN TJAŠA
|-
| POHLEVEN ŠPELA||naslov||||14.03.||17.03.||21.03.||KEPIC LEA||RADOJKOVIĆ MARKO||DOLINAR ANA
|-
| KEPIC LEA||naslov||||14.03.||17.03.||21.03.||VRANKAR ANDREJ||BRATOVŠ ANDREJA||MUSTAR JERNEJ
|-
| KMETIČ MIRJAM||naslov||||14.03.||17.03.||21.03.||MARIĆ TAMARA||REMŠKAR MAJA||KOMAN KATRA
|-
| JARC VERONIKA||naslov||||14.03.||21.03.||28.03.||REMŠKAR MAJA||MUSTAR JERNEJ||KEPIC LEA
|-
| KOMAN KATRA||naslov||||14.03.||21.03.||28.03.||ČUPOVIĆ VANA||KARNER TAJA||KMETIČ MIRJAM
|-
| OGRIS IZA||naslov||||14.03.||21.03.||28.03.||KNAPIČ EVA||BRGLEZ ŽIVA||VRANKAR ANDREJ
|-
| KERIN INES||naslov||||14.03.||21.03.||28.03.||KARNER TAJA||ŠTEMBERGER ROK||KERT DOMINIK
|-
| VERBANČIČ JANA||naslov||||21.03.||28.03.||04.04.||ŠTOK ULA||ZOTTEL ALJA||KNAPIČ EVA
|-
| KNAPIČ EVA||naslov||||21.03.||28.03.||04.04.||ZALAR MATJA||POHLEVEN ŠPELA||LORBEK SARA
|-
| REMŽGAR ANA||naslov||||21.03.||28.03.||04.04.||BOTONJIĆ SANDI||LORBEK SARA||ČUPOVIĆ VANA
|-
| GRDADOLNIK MAJA||naslov||||21.03.||28.03.||04.04.||MOHAR MAŠA||REMŽGAR ANA||FRANKO NIK
|-
| JAVORŠEK KAJA||naslov||||28.03.||04.04.||11.04.||GEC KARMEN||MARIĆ TAMARA||RADOJKOVIĆ MARKO
|-
| BRATOVŠ ANDREJA||naslov||||28.03.||04.04.||11.04.||ZOTTEL ALJA||ČUPOVIĆ VANA||GRDADOLNIK MAJA
|-
| CRČEK MITJA||naslov||||28.03.||04.04.||11.04.||BOHNEC IVO||KMETIČ MIRJAM||BRATOVŠ ANDREJA
|-
| MARIĆ TAMARA||naslov||||28.03.||04.04.||11.04.||NAVODNIK URŠKA||GEC KARMEN||REMŠKAR MAJA
|-
| ŠTEMBERGER ROK||naslov||||04.04.||11.04.||18.04.||JAVORŠEK KAJA||VRANKAR ANDREJ||BOTONJIĆ SANDI
|-
| LORBEK SARA||naslov||||04.04.||11.04.||18.04.||POHLEVEN ŠPELA||KNAPIČ EVA||VENE ROK
|-
| REMŠKAR MAJA||naslov||||04.04.||11.04.||18.04.||KERIN INES||POVŠE KATJA||CRČEK MITJA
|-
| KARNER TAJA||naslov||||04.04.||11.04.||18.04.||REMŽGAR ANA||VERBANČIČ JANA||RODE URŠKA
|-
| RODE URŠKA||naslov||||24.04.||03.05.||09.05.||GRDADOLNIK MAJA||FRANKO NIK||MARIĆ TAMARA
|-
| RADOJKOVIĆ MARKO||naslov||||24.04.||03.05.||09.05.||FRANKO NIK||VENE ROK||POVŠE KATJA
|-
| VENE ROK||naslov||||24.04.||03.05.||09.05.||VERBANČIČ JANA||NAVODNIK URŠKA||ZALAR MATJA
|-
| AMBROŽIČ MATEVŽ||naslov||||24.04.||03.05.||09.05.||ŠTEMBERGER ROK||HROVAT KARMEN||BOHNEC IVO
|-
| HROVAT KARMEN||naslov||||04.05.||09.05.||16.05.||KERT DOMINIK||JARC VERONIKA||KARNER TAJA
|-
| FRANKO NIK||naslov||||04.05.||09.05.||16.05.||LORBEK SARA||KEPIC LEA||REMŽGAR ANA
|-
| NAVODNIK URŠKA||naslov||||04.05.||09.05.||16.05.||AMBROŽIČ MATEVŽ||ŠTOK ULA||ŠTEMBERGER ROK
|-
| BRGLEZ ŽIVA||naslov||||09.05.||16.05.||23.05.||DOLINAR ANA||BOTONJIĆ SANDI||JARC VERONIKA
|-
| ČUPOVIĆ VANA||naslov||||09.05.||16.05.||23.05.||KOMAN KATRA||OGRIS IZA||NAVODNIK URŠKA
|-
| KERT DOMINIK||naslov||||09.05.||16.05.||23.05.||GORIČAN TJAŠA||GRDADOLNIK MAJA||RAUTER URŠKA
|-
| POVŠE KATJA||naslov||||16.05.||23.05.||30.05.||JARC VERONIKA||AMBROŽIČ MATEVŽ||BRGLEZ ŽIVA
|-
| GEC KARMEN||naslov||||16.05.||23.05.||30.05.||POVŠE KATJA||ZALAR MATJA||AMBROŽIČ MATEVŽ
|-
| GORIČAN TJAŠA||naslov||||16.05.||23.05.||30.05.||KMETIČ MIRJAM||RODE URŠKA||POHLEVEN ŠPELA
|-
| BOHNEC IVO||naslov||||23.05.||30.05.||06.06.||CRČEK MITJA||GORIČAN TJAŠA||ŠTOK ULA
|-
| ŠTOK ULA||naslov||||23.05.||30.05.||06.06.||BRGLEZ ŽIVA||DOLINAR ANA||KERIN INES
|-
| MUSTAR JERNEJ||naslov||||23.05.||30.05.||06.06.||BRATOVŠ ANDREJA||KOMAN KATRA||GEC KARMEN
|}

==Naloga==
* samostojno pripraviti seminar, katerega osnova je znanstveni članek s področja biokemije, ki ga po želji izberete v reviji s področja biokemije, ki ima faktor vpliva večji kot 3 in je bil objavljen v letu 2011. Poleg tega članka morate za seminar uporabiti še najmanj pet drugih virov! http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR
* osnovni članek in naslov pošljete meni, najkasneje pet dni pred rokom za oddajo (rok-5), da ocenim, če je primeren za predstavitev. Naslov vpišete v tabelo, takoj ko ste si ga izbrali!
* [[BIO1 Povzetki seminarjev|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom. Povezave do slik so dobrodošle, niso pa nujne.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 1800 do 2000 besed), vsebovati mora najmanj eno sliko. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Natisnjen seminar oddajte do roka vsakemu od recenzentov (docentu ga pošljite po e-pošti v formatu .doc ali .docx).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela, v predpisanem formatu elektronskega obrazca na internetu.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 15 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava- 5 minut. Recenzenti podajo oceno predstavitve in postavijo vsak vsaj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek na wikiju morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [[https://spreadsheets.google.com/viewform?formkey=dFM2SktfM3Q4VU1wNUQzdU45OTlWVXc6MA recenzentsko poročilo]] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://spreadsheets.google.com/viewform?formkey=dFd3TGhLV3ZSa2xsLVlmMVVUaEFURWc6MA mnenje] najkasneje v treh dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.google.com/url?sa=t&source=web&cd=6&sqi=2&ved=0CEUQFjAF&url=http%3A%2F%2Fwww.tre.sik.si%2Fmain%2Fpomoc%2Ffiles%2Fcitiranje_in_navajanje_virov.pdf&rct=j&q=citiranje%20po%20pravilniku%20ISO%20690&ei=jPBqTe6FC9DKswaWk-TmDA&usg=AFQjCNF8r6X9Y781sanDObaXNdCew4suUg&sig2=cTqKObSJsTicekWGRGa72g&cad=rja Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO1 Povzetki seminarjev 2011

2011-02-28T09:09:38Z

MatjaZalar: /* Ime in priimek: Naslov sdčkfjlsadjfkljsad fjasd seminarja */

== Ime in priimek: Naslov sdčkfjlsadjfkljsad fjasd test testklsnklaf sdlk fglkasdjklg ==
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam vel gravida urna. Nunc dignissim, augue eu pharetra volutpat, nisi neque mattis leo, sed rhoncus sem purus eget est. Etiam bibendum mi sit amet augue volutpat viverra. Sed ac nibh eu risus pellentesque commodo eget non odio. Vivamus nec odio vel felis tristique ultricies. Morbi sed mauris non est congue adipiscing. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean at ante ut arcu pretium mollis. Ut quis quam ut lacus auctor auctor. Pellentesque lobortis sagittis dolor ac gravida. Nulla in tellus dolor, a malesuada risus. Suspendisse ornare, mi in molestie gravida, velit sapien dapibus mauris, id ultrices velit libero id sapien. Sed pharetra dictum lectus in egestas.
Phasellus tempor, arcu a venenatis faucibus, orci arcu imperdiet mauris, quis adipiscing quam lectus vel dolor. Vestibulum sagittis ante quis ligula ullamcorper eget convallis justo fringilla. Mauris eget tellus at ante vulputate fermentum. Mauris placerat, arcu eu lobortis facilisis, neque dui pellentesque sapien, sit amet rutrum neque nisl vitae turpis. Donec urna elit, imperdiet nec tempus in, lacinia vitae ligula. Integer commodo, dolor non semper egestas, magna ipsum imperdiet quam, a lobortis purus elit bibendum mi. Cras in tortor non mauris pulvinar egestas.

== Ime in priimek: Naslov mojega seminarja ==
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam vel gravida urna. Nunc dignissim, augue eu pharetra volutpat, nisi neque mattis leo, sed rhoncus sem purus eget est. Etiam bibendum mi sit amet augue volutpat viverra. Sed ac nibh eu risus pellentesque commodo eget non odio. Vivamus nec odio vel felis tristique ultricies. Morbi sed mauris non est congue adipiscing. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean at ante ut arcu pretium mollis. Ut quis quam ut lacus auctor auctor. Pellentesque lobortis sagittis dolor ac gravida. Nulla in tellus dolor, a malesuada risus. Suspendisse ornare, mi in molestie gravida, velit sapien dapibus mauris, id ultrices velit libero id sapien. Sed pharetra dictum lectus in egestas.
Phasellus tempor, arcu a venenatis faucibus, orci arcu imperdiet mauris, quis adipiscing quam lectus vel dolor. Vestibulum sagittis ante quis ligula ullamcorper eget convallis justo fringilla. Mauris eget tellus at ante vulputate fermentum. Mauris placerat, arcu eu lobortis facilisis, neque dui pellentesque sapien, sit amet rutrum neque nisl vitae turpis. Donec urna elit, imperdiet nec tempus in, lacinia vitae ligula. Integer commodo, dolor non semper egestas, magna ipsum imperdiet quam, a lobortis purus elit bibendum mi. Cras in tortor non mauris pulvinar egestas.

== Ime in priimek: Naslov mojega seminarja ==
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam vel gravida urna. Nunc dignissim, augue eu pharetra volutpat, nisi neque mattis leo, sed rhoncus sem purus eget est. Etiam bibendum mi sit amet augue volutpat viverra. Sed ac nibh eu risus pellentesque commodo eget non odio. Vivamus nec odio vel felis tristique ultricies. Morbi sed mauris non est congue adipiscing. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean at ante ut arcu pretium mollis. Ut quis quam ut lacus auctor auctor. Pellentesque lobortis sagittis dolor ac gravida. Nulla in tellus dolor, a malesuada risus. Suspendisse ornare, mi in molestie gravida, velit sapien dapibus mauris, id ultrices velit libero id sapien. Sed pharetra dictum lectus in egestas.
Phasellus tempor, arcu a venenatis faucibus, orci arcu imperdiet mauris, quis adipiscing quam lectus vel dolor. Vestibulum sagittis ante quis ligula ullamcorper eget convallis justo fringilla. Mauris eget tellus at ante vulputate fermentum. Mauris placerat, arcu eu lobortis facilisis, neque dui pellentesque sapien, sit amet rutrum neque nisl vitae turpis. Donec urna elit, imperdiet nec tempus in, lacinia vitae ligula. Integer commodo, dolor non semper egestas, magna ipsum imperdiet quam, a lobortis purus elit bibendum mi. Cras in tortor non mauris pulvinar egestas.